Method for selecting a target nucleic acid sequence

ABSTRACT

The present invention relates to a method of selecting a target region of interest (ROI) in a target nucleic acid molecule using a nucleic acid probe comprising sequences capable of directing the cleavage of a target nucleic acid molecule to release a fragment comprising the ROI and sequences capable of templating the circularisation and ligation of the target fragment. The circularised molecule thus obtained contains the selected ROI and may be subjected to further analysis and/or amplification etc. Also provided are probes and kits for use in such methods.

The present invention relates to a method for selecting a target nucleicacid sequence. In particular, the present invention relates to a methodof selecting a target region of interest (ROI) in a target nucleic acidmolecule using a particular nucleic acid probe comprising sequencescapable of directing the cleavage of a target nucleic acid molecule torelease a fragment comprising the ROI and sequences capable oftemplating the circularisation and ligation of the target fragment. Thecircularised molecule thus obtained contains the selected ROI and may besubjected to further analysis and/or amplification etc. The selectionmethod of the invention thus provides a method not only for selectivelyisolating or separating a desired target sequence (ROI), but also fordetecting a target nucleic acid sequence (ROI), or for amplifying atarget sequence (ROI).

There are several methods described for selection and subsequentamplification of selected parts of a nucleic acid. Examples includeso-called selector probes (U.S. Pat. No. 7,883,849, or generalcircularisation of genomic fragments as described in Drmanac et al 2010.Science 327, 78-81 and U.S. Pat. No. 8,518,640).

Selector probes as described in U.S. Pat. No. 7,883,849 are designed tobind in a sequence-specific manner to a desired target sequence henceallowing it to be “selected” from a nucleic acid molecule, or indeedfrom a sample containing nucleic acid molecules. In the method of U.S.Pat. No. 7,883,849 partially double-stranded selector probes (either asingle symmetrical molecule in which the longer strand overhangs at bothends, or two asymmetrical molecules each having a single-strandedoverhang at only one end) are hybridised via their single-strandedoverhangs in a target-specific manner to both ends of single-stranded(denatured) target fragments resulting from fragmentation of the nucleicacid sample. In a particular embodiment of the method using thesymmetrical selector probe, only one end of the target fragmenthybridises to an end of the selector probe, the other end of theselector probe hybridising internally of the target nucleic acidfragment and requiring a structure-specific endonuclease to resolve theresulting structure by cleaving off the portion of the target fragmentprotruding beyond the internal hybridised region. In all cases,therefore, the selected portion of the target fragment is delineated bythe regions (whether both end regions or one end and one internalregion) of known sequence to which the selector probe(s) has beendesigned to hybridise. Following hybridisation (and, where appropriate,resolution of the secondary structure) the selector(s) and targetnucleic acid fragment are joined by ligation to give (i) in the case ofthe symmetrical selector probe, a circular nucleic acid molecule and(ii) in the case of the two asymmetrical selectors a linear moleculecomprising the target fragment flanked by selector probe sequences. Thedouble-stranded region of the selector probe(s) contains a primer pairmotif which is common to the plurality of different target-specificselectors used in a multiplex assay. Hence, amplification of multipletarget fragments can be achieved simultaneously whilst avoidingamplification artefacts which can result from the use of multiple,different primer pairs.

A particular problem identified in the selection methods of U.S. Pat.No. 7,883,849 is the requirement to carefully select which restrictionenzymes are used to digest a target nucleic acid molecule prior toselection, in order to avoid selecting enzymes which can cleave a targetnucleic acid molecule within the target sequence to be interrogated.This places limits on the degree of multiplexing that is possibleaccording to this method, and can lead to the amplification of undulylong nucleic acid fragments, thereby increasing the cost of analysingthe target nucleic acids selected.

In contrast to methods disclosed in the prior art, the present inventiondoes not require the prior cleavage of a sample nucleic acid prior toselection (although this is not precluded), and in particular it doesnot require cleavage in a manner to create specific binding sites forthe probe in the target molecule. Instead, it directs cleavage toparticular sites within a single-stranded target nucleic acid moleculewhere a nucleic acid probe binds, thereby circumventing the preciseselection of restriction enzymes to be used. Thus the present inventionprovides an improved method for the selection of target nucleic acidmolecules, and allows for the more precise selection of which sequencesare to be interrogated or analysed, e.g. by sequencing, but withoutrequiring target fragments containing specific probe binding sites attheir ends.

The invention accordingly provides a new kind of probe for selecting adesired or target nucleic acid sequence, which provides a new way ofgenerating a circular molecule containing the target sequence. Circularmolecules may readily be separated and handled (e.g. by digesting anylinear non-circularised nucleic acid molecules using exonucleaseenzymes) and may also be readily amplified and/or detected using rollingcircle amplification (RCA) or other amplification procedures. They arethus a very convenient way of providing a selected target sequence forfurther handling or processing, or analysis or detection etc.

The new probe for use according to the invention is a single strandednucleic acid molecule (i.e. an oligonucleotide) comprising fourtarget-specific binding sites arranged in order, the two “outer” bindingsites binding to complementary regions (or binding sites) in the targetmolecule which creates cleavage sites flanking the target sequence, orROI. The two inner target-binding sites of the probe serve as templatesfor circularisation of the fragment which is released from the targetmolecule by cleavage at the created cleavage sites. Sequence elementscan be placed adjacent to, or in between, the four target-binding sitesto enable or facilitate various downstream applications, e.g. elementsserving as tag or detection or identification sequences or sequences forthe capture (e.g. immobilisation) or amplification of the targetsequence/ROI, e.g. detection or ID tags or motifs (e.g. barcodes etc.),binding sites for detection probes or primers or for amplificationprimers, or a capture (or “anchor”) sequence able to bind to acomplementary sequence or cognate binding partner, e.g. provided on asolid support.

The method of the invention allows the use of single stranded targetmolecules and target fragments without the need to know the end-sequenceof the target. However, whilst the need to create fragments withtarget-specific ends is avoided and the method does not require afragmentation step, it may be convenient and desirable to include aninitial fragmentation step in the method.

Advantageously, and in contrast to certain prior art methods, theoriginal target molecule is circularised and not a copy thereof. Thusthe template for a subsequent amplification will be the originalmolecule, which can reduce errors in sequencing or sequence analysis ofthe amplicon. This is particularly advantageous in the context ofamplification by RCA where the original circularised molecule is thetemplate for each round of amplification, i.e. each copy of thecircle—the likelihood of an error being introduced in every lap of theRCA is very low, and particularly so at the same nucleotideposition—this makes the method of the invention very attractive forsequence analysis, e.g. genotyping applications, as the error rate isalmost infinitesimally low. The method thus has particular utility inthe detection or identification of rare mutations and similarapplications.

In a first aspect the invention accordingly provides a method ofselecting a target region of interest (ROI) in a target nucleic acidmolecule, said method comprising:

-   -   (a) providing a probe comprising in the following order four        target-binding sites capable of hybridising to complementary        binding sites in the target molecule, which complementary        binding sites flank the target ROI, as follows:        -   (i) a first target-binding site, complementary to a first            outer flanking sequence flanking a first side of the ROI in            the target molecule;        -   (ii) a second target-binding site, complementary to a second            inner flanking sequence flanking the other side of the ROI            on the target molecule;        -   (iii) a third target-binding site, complementary to a first            inner flanking sequence flanking the first side of the ROI            in the target molecule;        -   (iv) a fourth target-binding site, complementary to a second            outer flanking sequence flanking the other side of the ROI            in the target molecule;        -   such that only one of the second and third binding sites are            able to hybridise to their respective complementary binding            site in the target molecule when said first, fourth and the            other of second and third binding sites have hybridised;        -   wherein the first and fourth binding sites comprise a            sequence capable of creating a cleavage site when hybridised            to the target molecule;    -   (b) contacting the probe with the target molecule and allowing        the first, fourth and one of the second and third target binding        sites to hybridise to their respective complementary binding        sites in the target molecule, wherein the target molecule is at        least partially single stranded including in the region        comprising the four complementary binding sites, such that when        said probe has bound a partially double-stranded construct is        created comprising a loop in the probe strand comprising the        second or third target binding site which did not hybridise to        the target molecule and a loop in the target molecule strand        comprising the ROI and the complementary binding site which is        complementary to the second or third target binding region of        the probe which did not hybridise;    -   (c) cleaving the probe/target molecule construct at the cleavage        sites created by hybridisation of the first and fourth        target-binding sites thereby to release a target fragment        comprising the ROI flanked by the first and second inner        flanking sequences which are complementary to the third and        second target binding regions of the probe, one of which is        hybridised to its complementary binding site in the cleaved        probe;    -   (d) allowing the second or third target-binding site of the        probe which did not hybridise in step (b) to hybridise to its        complementary binding site in the target fragment, thereby to        bring the ends of the target fragment into juxtaposition for        ligation, directly or indirectly, using the cleaved probe as        ligation template;    -   (e) ligating the ends of the target fragment directly or        indirectly to circularise the target fragment;    -   (f) amplifying or separating the circularised target fragment,        thereby to select the ROI.

In a further aspect there is provided a probe for use in the method ofthe invention. More particularly, in this further aspect the inventionprovides an oligonucleotide probe for selecting a target ROI in a targetnucleic acid molecule, said probe comprising in the following order fourtarget-binding sites capable of hybridising to complementary bindingsites in the target molecule, which complementary binding sites flankthe target ROI, as follows:

-   -   (i) a first target-binding site, complementary to a first outer        flanking sequence flanking a first side of the ROI in the target        molecule;    -   (ii) a second target-binding site, complementary to a second        inner flanking sequence flanking the other side of the ROI on        the target molecule;    -   (iii) a third target-binding site, complementary to a first        inner flanking sequence flanking the first side of the ROI in        the target molecule;    -   (iv) a fourth target-binding site, complementary to a second        outer flanking sequence flanking the other side of the ROI in        the target molecule;        such that only one of the second and third binding sites are        able to hybridise to their respective complementary binding site        in the target molecule when said first, fourth and the other of        second and third binding sites have hybridised; and wherein the        first and fourth binding sites comprise a sequence capable of        creating a cleavage site when hybridised to the target molecule.

It will be understood from the above the probe does not comprise asequence which is capable of hybridising to the ROI.

The probe binds to the target nucleic acid molecule in a selectivemanner, allowing a “selected” target fragment comprising the ROI andflanking sequences to be cleaved from the target molecule andsubsequently circularised by ligation of the ends of the fragment in aprobe-templated ligation.

The selective cleavage is achieved by providing the probes with targetbinding sites (binding sites 1 and 4) which bind to regions(“complementary binding sites”) flanking the ROI in the target molecule(the “outer flanking sequences” on either side of the ROI), creatingcleavages sites which flank the ROI. Thus there are two outer bindingsites (1 and 4) and two inner binding sites (2 and 3) in the probecorresponding to cognate outer and inner flanking sites in the targetmolecule, which flank the ROI. To enable the cleaved fragment containingthe ROI to be circularised, a ligation template is provided by anucleotide sequence in the probe comprising target-binding sites 2 and3, which bind to complementary binding sites in the target moleculewhich flank the ROI inside the outer flanking sites (which create thecleavage sites). These complementary binding sites in the targetmolecule thus represent the ends of the fragment which is created by thecleavage, and their hybridisation to both the target binding sites 2 and3 in the probe following cleavage brings the fragment ends intojuxtaposition for ligation to circularise the fragment. In order forthis to happen, the order of binding sites 2 and 3 in the probe is“reversed” with respect to the order in which the complementary bindingsites appear in the target molecule—thus where binding site 1 binds tothe outer flanking region on a first side of the ROI, binding site 2binds to the inner flanking region on the other side of the ROI. Bindingsite 3 binds to the inner flanking region on the first side of the ROIand binding site 4 binds to the outer flanking on the other (second)side. This means that only one of binding sites 2 and 3 is able tohybridise to the target molecule when binding sites 1 and 4 arehybridised. In this way probe hybridisation causes the binding site 2 or3 which has not hybridised to the target to loop out. Similarly thecognate complementary binding site in the target molecule (complementaryto binding site 3 or 3 which has not bound) is caused to loop out.Further, since the probe does not comprise a sequence complementary tothe ROI, this also loops out. Probe hybridisation thus results in apartially double-stranded construct comprising an unhybridised singlestranded region (loop) in the probe strand comprising the unhybridisedbinding site 2 or 3, and a loop in the target strand comprising the ROIand the complementary binding site cognate to the unhybridised bindingsite 2 or 3 of the probe.

Thus three out of the four binding sites in the probe hybridise in theinitial probe binding step, including the two outer binding sites. Thefourth (inner) binding site is able to hybridise when it is releasedfrom the probe in the cleavage step, causing the ends of the cleavedfragment to be brought into juxtaposition for ligation. As mentionedabove, the ligation may be direct, when the fragment ends are ligateddirectly together, or it may be indirect when the two fragment endshybridise to their respective binding sites 2 and 3 in the probe with aspace (i.e. gap) or intervening sequence between them. As will bedescribed in more detail below, this may occur when binding sites 2 and3 are not immediately adjacent in the probe, but are separated by anintervening sequence. In such a configuration the gap between thehybridised fragment ends may be filled, either by a “gap”oligonucleotide, which hybridises to the intervening sequence betweenbinding sites 2 and 3 in the probe, or by extension of the hybridised 3′end of the fragment. The gap oligonucleotide may be providedpre-hybridised to the intervening sequence in the probe, or addedseparately, e.g. later during the method. It may also be provided in oneor more parts.

The orientation of the probe is not critical and the probe may be ineither orientation. Thus the binding sites 1, 2, 3 and 4 may lie 3′ to5′ in the probe or 5′ to 3′.

Further, the order of hybridisation of the three binding sites whichhybridise in step (b) is not critical and the binding sites mayhybridise simultaneously, or substantially simultaneously, and/orsequentially, in any order. In a representative embodiment, as shown inFIG. 3, binding site 2 does not hybridise in step (b) and hybridisesafter cleavage. However, in an alternative embodiment, binding site 2may hybridise in step (b) and binding site 3 may hybridise aftercleavage. The target ROI which is selected may be any desired sequenceor subsequence in a target nucleic acid molecule. The ROI may thusalternatively be termed a “target sequence” in a target nucleic acidmolecule. For example it may be a region of a nucleic acid in a samplewhich it is desired to amplify.

The term “selecting” is used broadly herein and includes any means ofselecting, isolating and/or separating a nucleic acid sequence ofinterest, for example from a nucleic acid sample which contains othernucleic acid molecules, particularly other DNAs, in addition to thetarget nucleic acid molecule or indeed from a longer nucleic acidmolecule containing the target ROI.

The target nucleic acid molecule is thus any nucleic acid moleculecontaining the target ROI. As will be discussed in more detail below, itmay thus be a genomic molecule or a fragment thereof, or any kind ofsynthetic or artificial nucleic acid molecule. Thus “selecting”encompasses any means of practically, if not actually physically,“separating” the target ROI from the other nucleic acids present in asample, and/or from the rest of the target molecule. The selected ROIcontained in the circularised target fragment may be subjected toamplification, e.g. by one of the many known methods of nucleic acidamplification, to amplify the ROI, for example for detection of the ROIor to enable further analysis, e.g. by sequencing, or to physicalseparation, e.g. capture, for example by immobilisation to a solidphase, optionally followed by amplification.

The method of the invention may thus include a further step of analysingthe circularised target fragment or an amplicon thereof. As will bedescribed further below, this may be by sequencing, or by a method ofsequence analysis (e.g. detecting the presence or absence of a sequencevariant or a particular nucleotide(s) in the ROI or determining themethylation status of the ROI), or by hybridisation of a detection probeto the ROI, optionally with further detection and/or signalamplification steps.

Such an analysis step will allow a target ROI to be detected, forexample in a sample containing nucleic acids. The target ROI maytherefore in one embodiment be a target analyte.

Accordingly, in a still further aspect the invention can also be seen toprovide a method of detecting a target ROI, for example using a probe ofthe invention to bind to a target nucleic acid molecule containing theROI and to select the target ROI as hereinbefore described.

The term “detecting” is also used broadly herein and includes any meansof identifying, detecting or determining or assaying for the presence ofthe target ROI, or any means of analysing the target ROI. Directanalysis of the target ROI (i.e. sequencing of all or any part of thetarget ROI) is encompassed by the term “detecting”.

The method of the invention may be performed in “simplex” format toenrich for a single target ROI (i.e. a single species of target ROI,which will normally be present in many copies) or for a plurality oftarget ROIs which are sufficiently similar in sequence, or flanked bysufficiently similar sequences so as to be possible to select them usingthe same probe. In this context it will be seen that the term “single”as used in relation to the probe means single in the context of aparticular target ROI, namely that one probe (or more particularly onetype or species of probe) is used for each target ROI (i.e. a singleprobe per target ROI). It is clear from the above that “single” probemeans single species of probe and does not imply any limitation on theactual number of probe molecules used.

Alternatively, a plurality (i.e. a plurality of species) of probes maybe used in a “multiplex” format simultaneously to enrich for a pluralityof target ROIs, which may be in the same, or more typically, indifferent, target molecules. Hence, in such a latter aspect the methodas defined above is for selecting a plurality of target ROIs, wherein aplurality of probes is provided, each designed to select a differenttarget ROI. In such an embodiment each probe may have a different set oftarget-binding sites, i.e. the probes have differenttarget-specificities. In such a multiplex method, for each target ROI ofthe plurality (i.e. each different type or species of target ROI) asingle (i.e. in the sense of a single species of) probe may be used.Thus, a plurality of probes may be used, with a (different) probe foreach target ROI. Thus in one embodiment each probe has different targetbinding sites, whereby a plurality of different target nucleic acidmolecules (and thus a plurality of different target ROIs) may beselected. In another embodiment, different ROIs in the same targetmolecule may be selected. In a still further embodiment, the same probe(i.e. comprising a single set of target binding sites) may be used todetect a plurality of different target ROIs that might be present withinthe same target nucleic acid molecule derived from a plurality (variety)of different sources.

The term “plurality” as used herein means 2 or more (or at least 2),more particularly 3 or more (or at least 3), or 4, 5, 6, 8, 10, 15, 20,30, 50, 70 or 100 or more etc. In certain embodiments even highernumbers of probes may be used and very many different target ROIs may beselected, e.g. 500, 1,000, 2,000, 5,000 or 10,000 or more. For example,10, 100, 1,000 or 10,000 different probes may simultaneously be used todetect or enrich for, respectively, 10, 100, 1,000 or 10,000 differenttarget ROIs.

The target ROI may be any sequence it may be desired to detect, analyseor amplify, for example a nucleotide sequence or a nucleic acid orselected part thereof in a pool of nucleic acid molecules or nucleotidesequences, for example genomic nucleic acids, whether human or from anysource, from a transcriptome, or any other nucleic acid (e.g. organellenucleic acids, i.e. mitochondrial or plastid nucleic acids), whethernaturally occurring or synthetic. The target nucleic acid molecule maytherefore be any kind of nucleic acid molecule. Thus it may be DNA orRNA, or a modified variant thereof. Thus the nucleic acid may be made upof ribonucleotides and/or deoxyribonucleotides as well as syntheticnucleotides that are capable of participating in Watson-Crick type oranalogous base pair interactions. Thus the nucleic acid may be or maycomprise, e.g. bi-sulphite converted DNA, LNA, PNA or any otherderivative containing a non-nucleotide backbone. The target molecule orROI may thus be coding or non-coding DNA, for example genomic DNA or asub-fraction thereof, or may be derived from genomic DNA, e.g. a copy oramplicon thereof, or it may be cDNA or a sub-fraction thereof, or anamplicon or copy thereof etc. Alternatively, the ROI or target moleculemay be or may be derived from coding (i.e. pre-mRNA or mRNA) ornon-coding RNA sequences (such as tRNA, rRNA, snoRNA, miRNA, siRNA,snRNA, exRNA, piRNA and long ncRNA). The probe may similarly be composedof or may comprise any nucleic acid as detailed above.

The sequence of the target ROI may not be known, providing that thesequence of the regions flanking the target ROI are known in order tofacilitate the design of the probe, which must be able to hybridise tothe flanking regions as defined and explained above.

The size of the target ROI is not critical and may vary widely. Thus inone embodiment of the present invention the target ROI may be at least10 nucleotides, and preferably at least 15 nucleotides in length. Thusthe target ROI may be at least 20, 25, 30, 40, 50, 60, 70, 80 or 90nucleotides in length. It is also anticipated that the present methodmay be used to select a longer target ROI, for example where the targetROI is at least 100, 150, 200, 300, or 400 nucleotides in length, or upto 500, 1,000, 1,500, 2,000, 2,500, 3,000, 5,000, 10,000, 25,000, 50,000or 100,000 nucleotides in length. Although RCA of nucleic acid circlesmay become less efficient as the size of the circle increases (e.g.above 5,000 or 10,000 nucleotides) it is still feasible. Thusrepresentative ranges of ROI length include from any one of 10, 12, or15 up to any one of 100,000, 50,000, 10,000, 5,000, 2,000, 1,000, 800,750, 700, 600 or 500 nucleotides. In particular embodiments, the sizerange may be from any one of 10, 12, or 15 to any one of 500, 400, 300,200, 100 or 50 nucleotides.

The target nucleic acid molecule may be present within a sample. Thesample may be any sample which contains any amount of nucleic acid, fromany source or of any origin, from which it is desired to select a targetROI. A sample may thus be any clinical or non-clinical sample, and maybe any biological, clinical or environmental sample in which the targetnucleic acid molecule may occur. More particularly, the sample may beany sample that contains nucleic acid. The target nucleic acid moleculemay occur in single-stranded or partially single-stranded or indouble-stranded form. However, as noted above for the practice of themethod the target molecule needs to be single-stranded at least in theregions where the probe hybridises. Where necessary, the method maytherefore comprise a step of rendering the target nucleic acid at leastpartially single-stranded, as discussed further below.

In one embodiment of the above method, the target ROI may be detected insitu, as it naturally occurs in the nucleic acid molecule in the sample.In such an embodiment the target nucleic acid molecule may be present ina sample at a fixed, detectable or visualisable position in the sample.The sample will thus be any sample which reflects the normal or native(“in situ”) localisation of the target nucleic acid molecule, i.e. anysample in which it normally or natively occurs. Such a sample willadvantageously be a cell or tissue sample. Particularly preferred aresamples such as cultured or harvested or biopsied cell or tissue samplesin which the target ROI may be detected to reveal the localisation ofthe target ROI relative to other features of the sample. As well as cellor tissue preparations, such samples may also include, for example,dehydrated or fixed biological fluids, and nuclear material such aschromosome/chromatin preparations, e.g. on microscope slides. Thesamples may be freshly prepared or they may be prior-treated in anyconvenient way such as by fixation or freezing. Accordingly, fresh,frozen or fixed cells or tissues may be used, e.g. FFPE tissue (FormalinFixed Paraffin Embedded).

Thus, representative samples may include any material which may containa target nucleic acid molecule, including for example foods and alliedproducts, clinical and environmental samples etc. The sample may be abiological sample, which may contain any viral or cellular material,including all prokaryotic or eukaryotic cells, viruses, bacteriophages,mycoplasmas, protoplasts and organelles. Such biological material maythus comprise all types of mammalian and non-mammalian animal cells,plant cells, algae including blue-green algae, fungi, bacteria, protozoaetc. Representative samples thus include clinical samples, e.g. wholeblood and blood-derived products such as plasma, serum and buffy coat,blood cells, other circulating cells (e.g. circulating tumour cells),urine, faeces, cerebrospinal fluid or any other body fluids (e.g.respiratory secretions, saliva, milk, etc.), tissues, biopsies, as wellas other samples such as cell cultures, cell suspensions, conditionedmedia or other samples of cell culture constituents, etc.

Although the method of the present invention may be used to select atarget ROI in a target nucleic acid molecule in an in situ (i.e. anative) setting, it is also contemplated that the method may be employedto select a target ROI in a target nucleic acid molecule in an in vitrodetection system, i.e. where a target nucleic acid molecule has beenisolated or purified from its native setting. The sample may thus be adirect product of a nucleic acid isolation procedure, or of a cell lysisprocedure, or it may further be fractionated or purified in some way,e.g. it may contain nucleic acids which have been partially or fullyseparated. The sample may also be treated in any way, e.g. the cDNAreverse transcript of an RNA molecule.

Although a fragmentation step is not necessary, it may in the case ofcertain target nucleic acid molecules or certain samples, e.g. in thecontext of genomic DNA, be desirable or convenient to include afragmentation step in the method, such that the nucleic acid in thesample, or the target molecule, is fragmented prior to the hybridisationof the probe. This may occur prior to or at the same time, orsubstantially the same time, as contacting the sample, or the targetnucleic acid molecule with the probe. As mentioned above, where thetarget molecule is double-stranded, a step of rendering the molecule atleast partially single-stranded is required. This step may be separateto the fragmentation step, e.g. after fragmentation, but may occur aspart of the fragmentation step. Fragmentation may be required or helpfulin order to allow the at least partially single-stranded nucleic acid tobe prepared.

The term “fragmenting” is used broadly herein to include any means bywhich the nucleic acid in the sample, or more particularly the targetmolecule, may be fragmented or cleaved. Thus, fragmentation may becarried out enzymatically, e.g. using restriction or other endonucleasesor nucleases such as DNase, and/or physically, e.g. by nebulisation orsonication or any shear-based methods. Such physical methods result inunpredictable, non-sequence-specific fragmentation, as do certain(non-restriction) endonucleases. Thus both random, and pre-determined(or site-specific) fragmentation is encompassed, but the latter is notnecessary. Also encompassed by “fragmenting” is fragmentation of anucleic acid sample which inherently may occur as a result of the age ofa sample, the conditions in which it is stored and any treatment of thesample (e.g. fixation, such as in formalin-fixed paraffin-embeddedsamples), and the degradation to which these factors contribute. Anysuitable class of restriction endonuclease may be used, including typeII and type IIs enzymes. Alternatively, fragmenting may be achievedusing a flap endonuclease (FEN), wherein an added nucleic acid oroligonucleotide is used to create a structure which is a substrate forsuch as structure-specific endonuclease, i.e. a structure having aprotruding non-hybridised 5′ end region. Fragmenting means may be usedin combination, e.g. the use together of two or more endonucleases, moreparticularly two or more restriction endonucleases, or the use togetherof an enzymatic and a physical means. Furthermore, the nucleic acidsample may be differently fragmented in separate aliquots, whichaliquots are then pooled and together subjected to the remaining stepsof the method of the invention. In certain cases, it may be appropriateand sufficient to fragment using a single restriction endonuclease, butin other cases the use of additional restriction endonucleases may bepreferred.

Hence, the fragmenting may be achieved by separating the nucleic acidsample into a plurality of aliquots and fragmenting the respectivealiquots with different means or different combinations of means, suchmeans being for example restriction enzymes. Any number of aliquots ofthe sample may be differently treated, e.g. 2 or more, or 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 or 20 or more etc. The aliquots are thensubjected to the remaining steps of the method and may be pooled forexample before step (b).

Before the target molecule can hybridise with the probe, it must be atleast partially single-stranded. This may be achieved, if necessary, byany means known in the art, such as denaturation, e.g. by heat or pH, orthrough the use of chemicals, e.g. alkali. Heat denaturation ispreferred.

Thus, after or concomitantly with any fragmenting step, the nucleic acidin the sample, including the target nucleic acid molecule, may ifnecessary be rendered at least partially single-stranded, to allow probehybridisation in step (b) to occur. Where the nucleic acid molecule isnot made completely single-stranded, it is required that it issingle-stranded at least the portions which comprise theprobe-complementary portions (so as to allow binding to the probe). Aswell as by denaturation, at least partial single-strandedness can beachieved by 3′ or 5′ exonucleolysis using an appropriate 3′ or 5′exonuclease. Starting at a free double-stranded fragment end, suchenzymes progressively degrade or digest one strand of a double-strandednucleic acid, leaving the complementary strand and rendering the nucleicacid single-stranded along the length of the enzyme's action. The extentof exonucleolytic degradation (i.e. the length of the resultingsingle-stranded region) may be controlled by the duration of thereaction. The duration of the exonuclease reaction is chosen in orderthat an appropriate length of one end of the strands of the fragments isremoved. The extent of digestion must be sufficient to allowhybridisation with the probe. Suitable exonucleases are known in the artand include, e.g. exonuclease III (3′) and lambda exonuclease (5′).

Further, in certain applications, for example in situ procedures asshown in FIG. 6, the duplex of a target nucleic acid molecule may beopened up to permit probe hybridisation, without fully denaturing thenucleic acid. Procedures for this are known in the art. In someembodiments, the circularised target fragment may be detected by RCA,wherein it may be useful to facilitate localisation of the RCA productto its native position, e.g. such that the RCA product functions as alocalised marker for the target nucleic acid molecule in the sample. Forinstance, the RCA product may be localised to the sample by immobilisingthe RCA product to the target nucleic acid or in proximity to the targetnucleic acid. Methods for immobilising the RCA product are describedfurther below and could be used to immobilise the RCA product in situ,e.g. wherein the nucleic acid molecule used to immobilise (e.g. captureor anchor) the circularised target fragment is immobilised to a specificlocation in the sample, e.g. the receptor or binding oligonucleotide,may consist of, or be attached to, an affinity binding molecule that iscapable of interacting with its cognate binding partner (e.g. the targetnucleic acid molecule), which is located in the sample, e.g. the cell.

The probe may therefore comprise a capture or anchor element, e.g. acapture or anchor sequence or an affinity binding group, that may beused to immobilise or localise the circularised fragment, or moreparticularly, an amplification product thereof, to a specific locatione.g. in a cell. Such a capture or anchor element may be provided in(e.g. as part of, or attached to) the second or third target-bindingsites of the probe, or an intervening sequence adjacent thereto, suchthat it is present in the portion of the probe which templates theligation of the target fragment comprising the ROI following cleavage ofthe probe. The capture or anchor element may bind to a molecule (i.e.its cognate binding partner) present in the sample (e.g. in the cell),including for example the target nucleic acid molecule itself. In oneembodiment, a capture or anchor sequence may hybridise to a portion orpart of the target nucleic acid molecule. Whilst this may not be thetarget ROI or a flanking region thereo, it may be a nucleic acidsequence adjacent or near to the ROI. Where the portion of the probewhich templates ligation comprises the capture or anchor sequence andalso acts as the primer for RCA, the amplification product may belocalised to the location of the target molecule. In particular, theamplification product may be bound (hybridised) to the target nucleicacid molecule by virtue of the capture or anchor sequence.

In some embodiments it is not necessary to actively immobilise the RCAproduct, e.g. to the target nucleic acid molecule, in order to produce alocalised signal in situ. In this respect, whilst not wishing to bebound by theory, it is hypothesized that the RCA product rapidly becomestoo large to readily diffuse away from its source, i.e. the RCA productis localised in its cell of origin even though it may not be directly orindirectly immobilized, e.g. to, or in proximity to, the target nucleicacid.

A number of different designs may be employed for the probe of thepresent invention. At its simplest, as depicted in FIGS. 1 and 3, theprobe is a linear molecule comprising four target binding sites capableof hybridising to complementary binding sites in the target molecule asdefined above, wherein the four target binding sites are immediatelyadjacent to one another. Thus the two outer binding sites 1 and 4 lie atthe ends of the probe.

In variant embodiments, the probe may comprise at least one (i.e. one,two, three or more) intervening sequence between any of the four targetbinding sites. One or more intervening sequences may be present betweenany two of the target binding sites within the probe, and multipleintervening sequences may thus be present within the probe, interspersedbetween the various target binding sequences. As noted above these maybe used to introduce elements useful for or which facilitate downstreamprocessing or handling, for example to introduce tag or detectionsequences or elements allowing the probe and/or circularised fragment tobe captured, for example for immobilisation to a solid phase. Thus theprobe may comprise an intervening sequence between the first and secondtarget binding sites, and/or an intervening sequence between the secondand third target binding sites, and/or an intervening sequence betweenthe third and fourth target binding sites. In a further embodiment, thenucleic acid probe may comprise intervening sequences between both thefirst and second target binding sites, and between the third and fourthtarget binding sites. Alternatively, the nucleic acid probe may compriseintervening sequences between any of the first, second, third and fourthtarget binding sites, such as between the first and second, and secondand third target binding sites; between the second and third, and thirdand fourth target binding sites; or between the first and second, secondand third, and third and fourth target binding sites. It is alsopossible for a probe to comprise more than one intervening sequencebetween any two target binding sites. The nucleic acid probe may alsocomprise additional sequences beyond the first and/or fourth targetbinding site. Importantly, neither the intervening sequences, nor theadditional sequences are capable of hybridising to the target nucleicacid molecule.

From the probe design it will be apparent that any intervening sequenceincorporated between binding sites 2 and 3 will result in there being agap between the hybridised ends of the cleaved target fragment. As notedabove, this gap needs to be filled in order for ligation of the ends totake place. Such an intervening sequence may therefore be used tointroduce a sequence into the circularised target molecule, for examplea tag or detection sequence, e.g. a barcode or identificatory motif, ora binding site for a detection probe or primer. Tags such asbarcodes/motifs or probe/primer binding sites may be designed withdifferent needs/purposes, for example to introduce a universal or commonsequence to enable different circularised target molecules in amultiplex setting to be processed together, e.g. to introduce a bindingsite for a universal or common amplification primer. This would enabledifferent circularised target fragments to be amplified together, e.g.in a library amplification by PCR or RCA. Alternatively or additionally,a tag/barcode sequence may be used to “label” different circularisedfragments so that they may readily be distinguished from one another(i.e. a “target” tag or marker), or to tag different samples etc., sothat they may be pooled prior to common/universal amplification together(i.e. a “sample” tag or marker). Thus, in a multiplex setting differentprobes (i.e. probes for different target ROIs) may be provided withdifferent tag sequences (e.g. different marker or detection sequences)and/or they may be provided with the same tag sequence(s), e.g. for theintroduction of a common or universal sequence.

As mentioned above, gap-filling may take place either by extending thehybridised 3′ end of the target fragment, or more typically byhybridising one or more gap oligonucleotides into the gap. The gapoligonucleotide may be provided as part of the probe (see e.g. FIG. 2B,2D or 4), e.g. prehybridised to the probe prior to contact with targetnucleic acid, or it may be provided at the same or substantially thesame time as contacting the probe with the target molecule, or it may beadded at any time afterwards, e.g. after probe hybridisation, or aftercleavage. The gap oligonucleotide may therefore be regarded as adetection, tag, barcode or ID motif oligonucleotide etc. As will bedescribed in more detail below and as depicted in FIG. 2D the gapoligonucleotide may comprise a region which is not complementary anddoes not hybridise to the intervening sequence between binding sites 2and 3 such that when it is hybridised it contains a non-hybridised loopinto which a tag, barcode, or ID motif sequence etc. may beincorporated.

In a further embodiment, capture or “anchor” sequence elements may beplaced between two binding sites, for example between binding sites 1and 2 and/or between binding sites 2 and 3, and/or between binding sites3 and 4. Such elements may be combined with detection/ID elementsbetween any of the sites. For example, capture elements between site 1and 2, 2 and 3, and 3 and 4 respectively may be combined with adetection/ID element between sites 2 and 3. Various representativepossibilities are depicted in FIG. 2. Such capture or anchor elementsmay be designed to hybridise to a cognate complementary binding site(i.e. a “bait” sequence), for example provided on a solid support, toenable the probe and/or target fragment to be immobilised. This may thusenable the method to be carried out on a solid phase for one or more ofthe steps, for example the probe may be immobilised before or afterhybridisation to the target molecule, or before or after the cleavagestep. This is depicted in more detail in FIG. 5.

Thus the intervening sequence(s) may contain or carry an element bywhich the target fragment may be detected or separated, e.g. identifiedor amplified or captured. By “contains or carries” is meant that such anelement may be contained within the nucleotide sequence of theoligonucleotide, e.g. a sequence tag (e.g. which can be used to identifya target fragment) or a probe or primer binding site or other nucleicacid-based affinity-binding site (for example a binding site for ahybridisation probe or for a DNA binding protein etc., which bindingsite may be viewed as a capture or detection element depending on thenature of the probe or affinity binding element, or a binding site for asequencing primer, which sequencing primer binding site may accordinglybe viewed as a detection element, or for an amplification primer, whichamplification primer binding site may accordingly be viewed as anamplification element), may be contained within the nucleotide sequenceof the oligonucleotide. Alternatively it may be attached or conjugatedor in any way linked or coupled to or associated with the interveningsequence. For example, it may be a functional moiety (e.g. a chemicalgroup or a molecule) which is attached etc. to the oligonucleotide, suchas an immobilisation moiety or a detection moiety (e.g. a reporter or alabel). An immobilisation moiety may, for example, be an affinitybinding moiety or group, e.g. one member of an affinity binding pair(i.e. an affinity ligand), which is attached or conjugated etc. to saidoligonucleotide, and is capable of binding to the other member of theaffinity binding pair (i.e. its cognate binding partner) for thepurposes of capture or separation, e.g. when the cognate binding partneris attached to a solid phase.

A detection element may, for example, include an identification element,namely an element which allows or permits identification, for example ofa particular target ROI, or of a sample (e.g. when samples are pooled),or indeed of an individual target nucleic acid molecule in the sample.Such an identification element or ID tag or motif may simply be asequence tag or motif, e.g. a particular or unique nucleotide sequence.This may thus be viewed as a sequence marker. The design of suchsequence tags/motifs or markers is well known in the art. For examplebarcode sequences/motifs for use as tags are widely used and known inthe art, for tagging samples or molecules etc. Degenerate sequences maybe used as the basis for such tags and again the use of degeneratesequence motifs in this way is known in the art.

A number of different tags may be included to mark or tag differentaspects of the target nucleic acid molecule or ROI. For example a tag,e.g. a barcode motif, may be included as a sample tag, together with atag for the particular target ROI for which the probe is designed to beselective. Advantageously, a “molecular” tag may be used to mark or tag(i.e. identify) an individual molecule in the sample, e.g. an originalmolecule of the sample. This can be particularly advantageous in thecontext of sequencing, and especially in NGS technologies, where it canbe valuable to track sequence reads back to an original, molecule whichis amplified for sequencing.

Thus, it will be seen that various types of identification element ortag may be used singly or in combination.

Sequence tag or barcodes can be in the region of 20 nucleotides, forexample from 7 to 30 nucleotides, e.g. 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides, in length. Sequence tags or barcodes can be randomlygenerated, for example a set of sequence tags may comprise tags with allpossible sequence combinations of the total number of nucleotides in thetag sequence.

Thus, the probe may contain a sample sequence tag, e.g. a feature whichallows a probe used in the method of the invention as performed on aparticular sample to be distinguished from a probe used in the method asperformed on a different sample and which thereby allows identificationof the sample from which a given target fragment has been circularised.

The sample tag allows identification of the sample from which aparticular target fragment and hence ROI originates. The samples may,for example, correspond to patient samples. If the method is performedin multiplex for detection of a number of different target ROIs fromdifferent samples, then separate sample and “target” tags may beincluded in each probe. Utilising the methods and probes of the presentinvention, this feature advantageously allows the pooling of samples,for example after contact with the probes and ligation of the probes totag each sample, different samples may be pooled.

A detection element may, as noted above, also be a binding sitecontained in the oligonucleotide sequence (e.g. a binding site for adetection probe or moiety or for a primer to be used in a detectionreaction, e.g. a sequencing primer) or it may be a detection moietywhich is carried in any way by the oligonucleotide, e.g. a reportergroup or moiety or a label, which may be directly or indirectlysignal-giving. For example it may a visualisable label, such as acoloured or fluorescent or particulate label (e.g. a magnetic orparamagnetic particulate label), or a moiety which contributes to ortakes part in a signal-giving reaction, e.g. an affinity binding partneror ligand or a substrate or co-factor for an enzyme.

A capture element may be any element for the amplification and/orcapture of the circularised target fragment. An “amplification element”may be used to amplify the circularised target fragment. Typically itwill be an amplification primer binding site. It may also be a bindingsite for one of a number or set (e.g. pair) of amplification primers,for example to allow exponential amplification, e.g. a PCR primer or aprimer for a PCR-based procedure. The primer binding site may also beused for the binding of a sequencing primer.

A “capture element” may be any moiety carried by (e.g. attached orconjugated to etc.) the probe (particularly the circularised targetfragment/probe construct), or any feature of the sequence of the probe(e.g. a binding site, intervening sequence), which may potentially beused selectively to attach a probe (particularly the circularised targetfragment/probe construct)) to a solid phase or support, including forexample a particle such as a bead. Hence, a capture element may beviewed as an “immobilisation element”. Numerous examples of suchelements are known in the art and include, e.g., an affinity bindingpartner, e.g. biotin or a hapten, capable of binding to its bindingpartner, i.e. a cognate binding partner, e.g. streptavidin or avidin, oran antibody, provided on the solid phase or support. A capture elementmay be a nucleotide sequence with complementarity to a corresponding“binding” or “receptor” oligonucleotide or nucleotide sequence providedon the solid support. Said interaction between the probe (particularlythe circularised target fragment/probe construct) and a solid phase(e.g. via an immobilised binding or receptor oligonucleotide) mayparticularly be mediated by click chemistry (Kolb H C et al, Angew ChemInt Ed Engl. 2001 Jun. 1; 40(11):2004-2021).

The solid phase may be any of the well-known supports or matrices whichare currently widely used or proposed for immobilisation, separationetc. These may take the form of particles (e.g. beads which may bemagnetic, paramagnetic or non-magnetic), sheets, gels, filters,membranes, fibres, capillaries, or microtitre strips, tubes, plates orwells etc. The support may be made of glass, silica, latex or apolymeric material. Suitable are materials presenting a high surfacearea for binding of the analyte. Such supports may have an irregularsurface and may be, e.g. porous or particulate, e.g. particles, fibres,webs, sinters or sieves. Particulate materials, e.g. beads, are usefuldue to their greater binding capacity, particularly polymeric beads.Conveniently, a particulate solid support used according to theinvention will comprise spherical beads. The size of the beads is notcritical, but they may, e.g. be of the order of diameter of at least 1and preferably at least 2 μm, and have a maximum diameter of preferablynot more than 10, and e.g. not more than 6 μm. Monodisperse particles,that is those which are substantially uniform in size (e.g. size havinga diameter standard deviation of less than 5%) have the advantage thatthey provide very uniform reproducibility of reaction. For example, toaid manipulation and separation, magnetic or paramagnetic beads areadvantageous. Other solid phases include very small particles which canefficiently contact a high proportion of the immobilisableoligonucleotides. Such particles may further be useful by retarding themovement of particle-attached target fragments through a gel, allowingseparation from free, non-particle-attached (non-target) fragments.Alternatively, also preferred is the use of a chromatographic matrixmodified with groups that can be reacted covalently or non-covalentlywith capture elements in the probe.

FIG. 4 depicts the operation of a probe containing an interveningsequence between binding sites 2 and 3, and comprising a hybridised tag(barcode) sequence.

As described above, and depicted particularly in FIG. 2D, the “gap”oligonucleotide which is hybridised to the intervening sequence betweenbinding sites 2 and 3 contains regions of complementarity to theintervening sequence, which regions are separated by a sequence that isnot complementary to the intervening sequence. Hence, the two regions ofcomplementarity to the intervening sequence are at the ends of thecomplementary oligonucleotide, and flank a region within theoligonucleotide that is not complementary to the intervening sequence.Consequently, the sequence that is not complementary to the interveningsequence forms a loop or bulge and may comprise a tag, for example abarcode sequence etc., e.g. for identifying a selected target ROI.

In all such embodiments described above the probe is linear and the twoouter binding sites (binding sites 1 and 4) lie at the ends of theprobe. Such a configuration represents one preferred embodiment of theinvention.

In a further embodiment, a probe may be provided which comprises ahairpin structure, and wherein it is necessary to first unfold the probeto open the loop before it can be used in the methods of the invention.The loop of the hairpin may contain one or more binding sites such thatthey are not available for hybridisation to the target molecule untilthe probe has been activated by opening, or unfolding, the hairpin toexpose, or release the binding site(s). For example binding sites 1 and2 may be contained in the loop, as depicted in FIG. 2E. Such a probedesign may be helpful in the preparation of large probe libraries. Ahairpin structure may also be known as a hairpin-loop or a stem-loop andthese terms are used interchangeably herein. A hairpin is anintra-molecular base-pairing pattern that can occur in a single-strandedDNA or RNA molecule. A hairpin occurs when two regions of the samestrand, usually complementary in nucleotide sequence when read inopposite directions, base-pair to form a double helix (a duplex) thatends in an unpaired, i.e. single-stranded, loop. The resulting structurecan be described as lollipop-shaped.

Thus in the present method a hairpin may be formed by the hybridisationof two regions of the probe. In a particular embodiment, the probe maycomprise one or more intervening sequences or additional sequences,which may allow the formation of a hairpin structure by hybridisingeither to one of the four binding sites within the probe, or byhybridising to another intervening sequence or additional sequencepresent in the probe. Advantageously the stem of the hairpin may beformed between an intervening sequence between binding sites 2 and 3 andan additional sequence provided at the end of binding site 1. In such anembodiment cleavage of the loop at or near to the junction betweenbinding site 1 and the additional sequence in the stem duplex mayrelease binding sites 1 and 2 and make them available for hybridisation,whilst retaining the additional sequence hybridised to the interveningsequence—the additional sequence thus forms a tag, detection, or barcodeoligonucleotide etc. (i.e. a gap oligonucleotide). This is depicted inFIG. 2D.

Depending on the exact nature of the hairpin probe design, the hairpinmay be unfolded or “opened” in various ways including by disruption ofthe duplex of the hairpin structure, or by cleavage, e.g. in or near theloop. As described above, it may be advantageous to retain the doublestranded element of the hairpin structure and to cleave the loop of thehairpin structure. A discussion of the techniques which may be employedto unfold a hairpin structure is provided in WO2012/152942, which isincorporated herein by reference in its entirety. As discussed below,cleavage is preferably enzymatic cleavage.

As mentioned above, unfolding may also be achieved by disrupting atleast part of the double stranded element of the hairpin structure. Thismay be achieved by altering the conditions of the sample such that thehairpin structure is no longer a thermodynamically favourable structure,e.g. by altering the temperature or salt concentrations of the solution.Similarly, the hairpin structure may be destabilised by modification ofone or more of the nucleotide bases in the duplex to disrupt thehydrogen bonds (so-called Watson-Crick base pairing) which anneal thetwo strands. For example, cleavage of the base from the nucleotide maybe sufficient to disrupt the duplex enough to “unfold” the hairpin.

Alternatively, the hairpin structure may be unfolded by out-competingthe double stranded element of the hairpin structure with“anti-blocking” oligonucleotides. For instance, in the presence of ahigh concentration of an anti-blocking oligonucleotide that iscomplementary to one of the strands of the hairpin structure, theinteraction (hybridization) between the anti-blocking oligonucleotideand the nucleic acid probe will be favoured over the hairpin structure.

Preferably, however, the hairpin is opened by cleaving the probe.

“Cleavage” is defined broadly herein to include any means of breaking ordisrupting a nucleotide chain (i.e. a nucleotide sequence). Cleavage maythus involve breaking a covalent bond. Typically cleavage will involvecleavage of nucleotide chain (i.e. strand cleavage or strand scission),for example by cleavage of a phosphodiester bond.

For instance, the hairpin structure may comprise or may be engineered ormodified to comprise a restriction endonuclease recognition sequence. Ina preferred embodiment, e.g. where the hairpin structure comprises arestriction endonuclease recognition site, the restriction endonucleasewill cleave only a single strand of the duplex portion of the hairpinstructure. For example, this may be achieved by hybridising anoligonucleotide (termed herein a “restriction oligonucleotide”) to thesingle-stranded loop of the hairpin structure to comprise a duplexwithin the loop. At least part of the formed duplex will comprise arestriction endonuclease recognition site, which can be cleavedresulting in unfolding of the hairpin structure. Any suitablerestriction endonuclease may be used to unfold the hairpin structure.

In some embodiments, the loop of the hairpin structure may comprise aregion of intramolecular complementarity such that it is able to form aduplex within the loop, i.e. the loop contains a double stranded region(a duplex) that forms a protrusion from the loop and is thereforedistinct from the “stem” of the hairpin structure. The internal duplexof the loop may comprise a cleavage site, e.g. a restrictionendonuclease recognition site, wherein cleavage of the duplex within theloop results in unfolding of the hairpin structure. Thus, in someembodiments, the additional sequence provided at the end of the firstbinding site may contain a region of intramolecular complementarity.

In yet further embodiments of the invention an exonuclease enzyme may beused to degrade one strand of the hairpin duplex, thereby releasing thesingle-stranded loop of the hairpin, i.e. unfolding the probe. Theexonuclease enzyme may have 5′ or 3′ exonuclease activity depending onthe orientation of the hairpin structure.

In other embodiments, cleavage may comprise breaking covalent bondswithin one or more nucleotides in a nucleic acid sequence. For example,where the hairpin structure comprises uracil residues, at least aportion of the duplex in the hairpin structure may be disrupted byremoving one or more uracil bases, i.e. cleavage of said bases from thenucleic acid using a uracil-DNA glycosylase enzyme. Removal of said oneor more uracil bases results in the loss of some hydrogen bonds betweenthe two strands of the hairpin duplex, resulting in a loss of stabilityand unfolding of the nucleic acid domain.

In some embodiments a cleavage site may be created by incorporating oneor more uracil residues into the loop sequence. In a particularlypreferred embodiment, the hairpin structure can be unfolded by treatmentwith a uracil-DNA glycosylase (UNG) enzyme in combination with anendonuclease enzyme capable of recognising apurinic/apyrimidinic (AP)sites of dsDNA, e.g. endonuclease IV.

In a further preferred embodiment the hairpin structure may be cleaved,and thereby unfolded, using a nickase enzyme, which cleaves only onestrand in the duplex of the hairpin structure. Nickases areendonucleases which cleave only a single strand of a DNA duplex. Asdescribed above, a cleavage site may be introduced in thesingle-stranded loop of the hairpin structure, e.g. by annealing(hybridising) and oligonucleotide to said loop.

Some nickases introduce single-stranded nicks only at particular siteson a DNA molecule, by binding to and recognizing a particular nucleotiderecognition sequence. A number of naturally-occurring nickases have beendiscovered, of which at present the sequence recognition properties havebeen determined for at least four. Nickases are described in U.S. Pat.No. 6,867,028, which is herein incorporated by reference in its entiretyand any suitable nickase may be used in the methods of the invention.

In some preferred embodiments that utilise a nickase enzyme, the nickaseenzyme is removed from the assay or inactivated following unfolding ofthe nucleic acid probe to prevent unwanted cleavage of ligationproducts.

As discussed above, the nucleic acid probe for use in the methods of theinvention comprises four target binding sites which are complementary tothe regions of the target nucleic acid molecule flanking the target ROI.“Complementarity” as used herein refers to functional complementarity,i.e. capable of mediating hybridisation, and need not refer to 100%complementarity between two nucleic acid molecules. Hybridisationaccording the present invention includes the formation of a duplexbetween nucleotide sequences which are sufficiently complementary toeach other, whether by Watson-Crick type base pairing or by anyanalogous base pairing. The hybridisation is a productive hybridisation,that is a hybridisation which is stable enough or strong enough for theprobe to be able to perform its function, e.g. for probe/target moleculehybrid to be separated from the sample, or for the cleavage sitescreated to be cleaved, and for the target fragment to be able to becircularised by ligation.

Thus, complementary nucleotide sequences will combine with specificityto form a stable duplex under appropriate hybridization conditions. Forinstance, two sequences are complementary when a section of a firstsequence can bind to a section of a second sequence in an anti-parallelsense wherein the 3′-end of each sequence binds to the 5′-end of theother sequence and each A, T(U), G and C of one sequence is then alignedwith a T(U), A, C and G, respectively, of the other sequence. RNAsequences can also include complementary G=U or U=G base pairs. Thus,two sequences need not have perfect homology to be “complementary” underthe invention. Usually two sequences are sufficiently complementary whenat least about 85% (preferably at least about 90%, and most preferablyat least about 95%) of the nucleotides share base pair organization overa defined length of the molecule.

It would be a matter of routine to the person skilled in this artappropriately to design the target binding sites in the probe, e.g.taking into account length of the sites, G/C content and Tm etc., andthe desired hybridisation pattern (i.e. which of binding sites 2 or 3hybridises initially to the probe). Typically the target binding site isat least 5 nucleotides long, more typically at least 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50 nucleotides in lengthor any integer between or up to or above any of these.

The cleavage in step (c) of the method may be achieved by any convenientmeans but is preferably enzymatic cleavage.

Thus, in a preferred embodiment, the cleavage sites created in thepartially double-stranded probe/target molecule hybrid represent acleavage recognition site, e.g. a sequence that is recognised by one ormore enzymes capable of cleaving nucleic acid molecules. The twocleavage sites created in step (c) may either be the same or different.

Any suitable cleavage enzyme may be used to cleave the partiallydouble-stranded construct to release a target fragment comprising theregion of interest flanked by the first and second inner flankingsequences which are complementary to the third and second bindingregions of the nucleic acid probe, one of which is hybridised to itscomplementary binding site in the cleaved probe.

In a preferred embodiment, the cleavage site is a restriction site. Inthis preferred embodiment the restriction endonuclease will cleave bothstrands of the cleavage site. The endonuclease may alternatively cleavea single strand, namely the target strand. By way of example, FIG. 7shows a representative probe and target molecule showing how a cleavagesite may be created and cleaved.

As discussed above, the second and third target binding sites templatethe ligation and thereby circularisation of the target fragment, wherebythe second or third target binding site that is not hybridised to thetarget nucleic acid molecule may hybridise to its complementary regionflanking the target ROI, thereby to bring the ends of the targetfragment into juxtaposition for ligation.

As discussed above, the ends of the target fragment may be positioneddirectly adjacent to each other where the second and third targetbinding sites are directly adjacent within the probe, and thus ligationmay take place directly between the ends of the target fragment.Alternatively, the ends of the target fragment may not be positioneddirectly adjacent to each other, i.e. where there is an interveningsequence between the second and third target binding sites in the probe.In such instances, the ends of the target nucleic acid molecule probemay be ligated indirectly, e.g. via one or more gap oligonucleotides orafter the “gap-fill” extension of the 3′ end of the oligonucleotide. Thegap oligonucleotide will be complementary to the intervening sequencebetween the second and third target binding sites, and may either beadded to the sample separately to the probe, or may be hybridised to thenucleic acid probe prior to the selection of the target ROI.

The ligation step may be performed by procedures well known in the art.Enzymes appropriate for the ligation step are known in the art andinclude, e.g. Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain9°N) DNA ligase (9°N™ DNA ligase, New England Biolabs), Ampligase™(Epicentre Biotechnologies) and T4 DNA ligase.

A target ROI circularised according to the method herein may be directlyseparated, analysed or detected, or may instead first be amplified.Indeed it may be detected by means of the amplification. Amplificationof the target ROI may be performed by any suitable method for amplifyingnucleic acids and in particular circular nucleic acids. Amplificationmay be linear or exponential, as desired, where representativeamplification protocols of interest include, but are not limited to:polymerase chain reaction (PCR); isothermal amplification,rolling-circle amplification (RCA), and their well-known variants, suchas hyperbranched RCA, etc. Other nucleic acid amplification methods mayinclude Loop mediated isothermal amplification (LAMP), SMartAmplification Process (SMAP), Nucleic acid sequence based amplification(NASBA), or ligase chain reaction (LCR). Where the detection stepincludes an amplification, the amplification product may be detected, todetect the target ROI.

Amplification methods based on RCA represent one preferred embodimentand in a particular aspect the amplification may involve a second roundof RCA, for example a superRCA (sRCA) reaction as described inWO2014/076209, herein incorporated by reference. In such a sRCA reactiona secondary RCA reaction is performed using a further RCA templatecircle and the RCA is primed by a primer which is hybridised to theprimary RCA product (here the amplicon of a first RCA step using thecircularised target fragment as template). The primer is provided insuch a way that it remains hybridised to the first RCA productthroughout the secondary RCA, such that the secondary RCA product islocalised to the primary RCA product. In a further preferred embodimentof a localised super RCA reaction, the secondary RCA reaction may betemplated by a padlock probe which hybridises to the primary RCA productand is ligated to form a circle which is then subjected to a secondaryRCA reaction (a so-called “Padlock sRCA” which is described in ourco-pending GB patent application No. 1320145.4, published asWO2015/071445).

In such sRCA reactions the secondary RCA product is unrelated to theprimary RCA template, i.e. the first circle, which in this case is thecircularised target fragment. Thus a sRCA is used as a means of signalamplification, e.g. in a detection method, rather than as means ofamplifying the target ROI in, e.g. a preparative method. For suchpurposes, a circle-to-circle RCA reaction (as described inWO/2003/012119) may be used to enhance the amount of product generatedby the RCA, which is essentially a linear amplification process, or ahyperbranched RCA.

Alternatively an exponential amplification reaction such as PCR may beused. Amplification of the circularised target fragment by PCRrepresents another preferred embodiment. As discussed above, bindingsites for PCR primers may conveniently be provided by a gapoligonucleotide incorporated into the circularised molecule.Alternatively, amplification primers may be designed to bind elsewherein the target fragment. In multiplex embodiments universal or common PCRprimer binding sites may be introduced and used to amplify differentcircularised target fragments in parallel using a single primer pair.Such use of a single primer pair has been demonstrated in other contextsand applications, e.g. for Selector probes as disclosed in U.S. Pat. No.7,883,849 as discussed above.

It will be apparent that the portions of the probe that template theligation of the target ROI (i.e. the second and third target nucleicacid binding sites) can remain bound to the target ROI after ligationand circularisation have taken place. Thus in one embodiment of thepresent invention, the second or third target binding site of thenucleic acid probe (depending on the orientation) may act as a primer toinitiate rolling circle amplification. In an alternative embodiment, aprimer may be added to the sample to initiate rolling circleamplification.

Although amplification of the circularised target fragment is convenientand in many applications of the method it may be preferred, it ispossible also to separate the circularised target fragment in otherways, for example by digesting any linear molecules present using anexonuclease, thereby to enrich for circular nucleic acids, or by othernucleic acid separation or fractionation procedures known in the art.

It is further apparent that the method outlined above can be used togenerate a circularised molecule comprising the target ROI in solution.Thus the method may be performed in a homogenous format. However in analternative embodiment the probe may be immobilised on a solid supportprior to, or after, hybridisation to the target nucleic acid molecule,or indeed at any stage of the method. Means for immobilising the probeor target fragment may be introduced by way of an intervening sequencebetween any two of its target binding sites. Such a method is depictedin FIG. 5.

As shown, the intervening sequence may be between the second and thirdtarget binding sites, and may be hybridised to a complementary nucleicacid molecule, thereby forming a partially double-stranded nucleic acidprobe. In a first embodiment the intervening sequence may be attached toa solid support. In a second embodiment the complementary nucleic acidmolecule can be attached to a solid support. In a further embodiment,the intervening sequence may be between the first and second, or betweenthe third and fourth target binding sites, and may be hybridised to acomplementary nucleic acid molecule, thereby forming a partiallydouble-stranded nucleic acid probe, and the complementary nucleic acidmolecule can be attached to a solid support.

The circularised target fragment which is selected according to themethod of the invention, or an amplicon thereof, may be detected. Thedetection may be by nucleic acid readout platform, including sequencingor any sequence-analysis procedure, real-time PCR and sRCA. A selectedand amplified target ROI can be detected or analysed using a number ofknown means, e.g. by hybridisation of a detection probe to the amplifiedproduct which may be labelled, or which may take part in furthersignal-giving or signal-amplification reaction, e.g. a ligation-basedreaction, e.g. a padlock probe, or a probe for an OLA assay. Padlockprobes may be detected in further amplification reactions, e.g. in asRCA reaction.

Thus the amplified products of an amplification reaction may be detectedusing any convenient protocol, which may detect the amplificationproducts non-specifically or specifically, as described in greaterdetail below. Representative non-specific detection protocols ofinterest include protocols that employ signal producing systems thatselectively detect double stranded DNA products, e.g., viaintercalation. Representative detectable molecules that find use in suchembodiments include fluorescent nucleic acid stains, such asphenanthridinium dyes, including monomers or homo- or heterodimersthereof, that give an enhanced fluorescence when complexed with nucleicacids. Examples of phenanthridinium dyes include ethidium homodimer,ethidium bromide, propidium iodide, and other alkyl-substitutedphenanthridinium dyes. In another embodiment of the invention, thenucleic acid stain is or incorporates an acridine dye, or a homo- orheterodimer thereof, such as acridine orange, acridine homodimer,ethidium-acridine heterodimer, or 9-amino-6-chloro-2-methoxyacridine. Inyet another embodiment of the invention, the nucleic acid stain is anindole or imidazole dye, such as Hoechst 33258, Hoechst 33342, Hoechst34580 (BIOPROBES 34, Molecular Probes, Inc. Eugene, Oreg., (May 2000))DAPI (4′,6-diamidino-2-phenylindole) or DIPI(4′,6-(diimidazolin-2-yl)-2-phenylindole). Other permitted nucleic acidstains include, but are not limited to, 7-aminoactinomycin D,hydroxystilbamidine, LDS 751, selected psoralens (furocoumarins), styryldyes, metal complexes such as ruthenium complexes, and transition metalcomplexes (incorporating Tb³⁺ and Eu³⁺, for example). In certainembodiments of the invention, the nucleic acid stain is a cyanine dye ora homo- or heterodimer of a cyanine dye that gives an enhancedfluorescence when associated with nucleic acids. Any of the dyesdescribed in U.S. Pat. No. 4,883,867 to Lee (1989), U.S. Pat. No.5,582,977 to Yue et al. (1996), U.S. Pat. No. 5,321,130 to Yue et al.(1994), and U.S. Pat. No. 5,410,030 to Yue et al. (1995) (all fourpatents incorporated by reference) may be used, including nucleic acidstains commercially available under the trademarks TOTO, BOBO, POPO,YOYO, TO-PRO, BO-PRO, PO-PRO and YO-PRO from Molecular Probes, Inc.,Eugene, Oreg. Any of the dyes described in U.S. Pat. No. 5,436,134 toHaugland et al. (1995), U.S. Pat. No. 5,658,751 to Yue et al. (1997),and U.S. Pat. No. 5,863,753 to Haugland et al. (1999) (all three patentsincorporated by reference) may be used, including nucleic acid stainscommercially available under the trademarks SYBR Green, EvaGreen, SYTO,SYTOX, PICOGREEN, OLIGREEN, and RIBOGREEN from Molecular Probes, Inc.,Eugene, Oreg. In yet other embodiments of the invention, the nucleicacid stain is a monomeric, homodimeric or heterodimeric cyanine dye thatincorporates an aza- or polyazabenzazolium heterocycle, such as anazabenzoxazole, azabenzimidazole, or azabenzothiazole, that gives anenhanced fluorescence when associated with nucleic acids, includingnucleic acid stains commercially available under the trademarks SYTO,SYTOX, JOJO, JO-PRO, LOLO, LO-PRO from Molecular Probes, Inc., Eugene,Oreg.

In yet other embodiments, a signal producing system that is specific forthe amplification product, as opposed to double stranded molecules ingeneral, may be employed to detect the amplification. In theseembodiments, the signal producing system may include a detection probethat specifically binds to a sequence found in the amplificationproduct, where the detection probe may be labelled with a directly orindirectly detectable label. A directly detectable label is one that canbe directly detected without the use of additional reagents, while anindirectly detectable label is one that is detectable by employing oneor more additional reagents, e.g., where the label is a member of asignal producing system made up of two or more components. In manyembodiments, the label is a directly detectable label, where directlydetectable labels of interest include, but are not limited to:fluorescent labels, radioisotopic labels, chemiluminescent labels, andthe like. In many embodiments, the label is a fluorescent label, wherethe labelling reagent employed in such embodiments is a fluorescentlytagged nucleotide(s), e.g. fluorescently tagged CTP (such as Cy3-CTP,Cy5-CTP) etc. Fluorescent moieties which may be used to tag nucleotidesfor producing labelled probes include, but are not limited to:fluorescein, the cyanine dyes, such as Cy3, Cy5, Alexa 555, Bodipy630/650, and the like. Other labels, such as those described above, mayalso be employed as are known in the art.

In certain embodiments, the specifically labelled detection probes arelabelled with “energy transfer” labels. As used herein, “energytransfer” refers to the process by which the fluorescence emission of afluorescent group is altered by a fluorescence-modifying group. If thefluorescence-modifying group is a quenching group, then the fluorescenceemission from the fluorescent group is attenuated (quenched). Energytransfer can occur through fluorescence resonance energy transfer, orthrough direct energy transfer. The exact energy transfer mechanisms inthese two cases are different. It is to be understood that any referenceto energy transfer in the instant application encompasses all of thesemechanistically-distinct phenomena. As used herein, “energy transferpair” refers to any two molecules that participate in energy transfer.Typically, one of the molecules acts as a fluorescent group, and theother acts as a fluorescence-modifying group. “Energy transfer pair” isused to refer to a group of molecules that form a single complex withinwhich energy transfer occurs. Such complexes may comprise, for example,two fluorescent groups which may be different from one another and onequenching group, two quenching groups and one fluorescent group, ormultiple fluorescent groups and multiple quenching groups. In caseswhere there are multiple fluorescent groups and/or multiple quenchinggroups, the individual groups may be different from one another. As usedherein, “fluorescence resonance energy transfer” or “FRET” refers to anenergy transfer phenomenon in which the light emitted by the excitedfluorescent group is absorbed at least partially by afluorescence-modifying group. If the fluorescence-modifying group is aquenching group, then that group can either radiate the absorbed lightas light of a different wavelength, or it can dissipate it as heat. FRETdepends on an overlap between the emission spectrum of the fluorescentgroup and the absorption spectrum of the quenching group. FRET alsodepends on the distance between the quenching group and the fluorescentgroup. Above a certain critical distance, the quenching group is unableto absorb the light emitted by the fluorescent group, or can do so onlypoorly. As used herein “direct energy transfer” refers to an energytransfer mechanism in which passage of a photon between the fluorescentgroup and the fluorescence-modifying group does not occur. Without beingbound by a single mechanism, it is believed that in direct energytransfer, the fluorescent group and the fluorescence-modifying groupinterfere with each others' electronic structure. If thefluorescence-modifying group is a quenching group, this will result inthe quenching group preventing the fluorescent group from even emittinglight.

The energy transfer labelled detection probe, e.g. oligonucleotide, maybe structured in a variety of different ways, so long as it includes adonor, acceptor and target nucleic acid binding domains. As such, theenergy transfer labelled oligonucleotides employed in these embodimentsof the method are nucleic acid detectors that include a fluorophoredomain where the fluorescent energy donor, i.e., donor, is positionedand an acceptor domain where the fluorescent energy acceptor, i.e.,acceptor, is positioned. As mentioned above, the donor domain includesthe donor fluorophore. The donor fluorophore may be positioned anywherein the nucleic acid detector, but is typically present at the 5′terminus of the detector. The acceptor domain includes the fluorescenceenergy acceptor. The acceptor may be positioned anywhere in the acceptordomain, but is typically present at the 3′ terminus of the nucleic aciddetector or probe.

In addition to the fluorophore and acceptor domains, the energy transferlabelled probe oligonucleotides also include a target nucleic acidbinding domain, which binds to a target nucleic acid sequence (e.g. atag, for example a barcode sequence) found in the amplification productof interest (as described above). Specific examples of such labelledoligonucleotide probes include the TaqMan® type probes, as described inU.S. Pat. No. 6,248,526, the disclosure of which is herein incorporatedby reference (as well as Held et al., Genome Res. (1996) 6:986-994;Holland et al., Proc. Natl Acad. Sci. USA (1991) 88:7276-7280; and Leeet al., Nuc. Acids Res. (1993) 21:3761-3766). Examples of other types ofprobe structures include: Scorpion probes (as described in Whitcombe etal., Nature Biotechnology (1999) 17:804-807; U.S. Pat. No. 6,326,145,the disclosure of which is herein incorporated by reference), Sunriseprobes (as described in Nazarenko et al., Nuc. Acids Res. (1997)25:2516-2521; U.S. Pat. No. 6,117,635, the disclosure of which is hereinincorporated by reference), Molecular Beacons (Tyagi et al., NatureBiotechnology (1996) 14:303-308; U.S. Pat. No. 5,989,823, the disclosureof which is incorporated herein by reference), and conformationallyassisted probes.

The next step in the subject methods is signal detection from thelabelled amplification products of interest, where signal detection mayvary depending on the particular signal producing system employed. Incertain embodiments, merely the presence or absence of detectablesignal, e.g., fluorescence, is determined and used in the subjectassays, e.g., to determine or identify the presence or absence of thetarget ROI. Depending on the particular label employed, detection of asignal may indicate the presence or absence of the target ROI.

In those embodiments where the signal producing system is a fluorescentsignal producing system, signal detection typically includes detecting achange in a fluorescent signal from the reaction mixture to obtain anassay result. In other words, any modulation in the fluorescent signalgenerated by the reaction mixture is assessed. The change may be anincrease or decrease in fluorescence, depending on the nature of thelabel employed, but in certain embodiments is an increase influorescence. The sample may be screened for an increase in fluorescenceusing any convenient means, e.g., a suitable fluorimeter, such as athermostable-cuvette or plate-reader fluorimeter, or where the sample isa tissue sample on a microscope slide, fluorescence may be detectedusing a fluorescence microscope. Fluorescence is suitably monitoredusing a known fluorimeter. The signals from these devices, for instancein the form of photo-multiplier voltages, are sent to a data processorboard and converted into a spectrum associated with each sample tube.Multiple tubes, for example 96 tubes, can be assessed at the same time.Thus, in some embodiments multiple targets may be detected in parallel,whereas in other embodiments multiple targets may be detectedsequentially.

Where the detection protocol is a real time protocol, e.g., as employedin real time PCR reaction protocols, data may be collected in this wayat frequent intervals, for example once every 3 minutes, throughout thereaction. By monitoring the fluorescence of the reactive molecule fromthe sample during each cycle, the progress of the amplification reactioncan be monitored in various ways. For example, the data provided bymelting peaks can be analysed, for example by calculating the area underthe melting peaks and these data plotted against the number of cycles.

The spectra generated in this way can be resolved, for example, using“fits” of pre-selected fluorescent moieties such as dyes, to form peaksrepresentative of each signalling moiety (i.e. fluorophore). The areasunder the peaks can be determined which represents the intensity valuefor each signal, and if required, expressed as quotients of each other.The differential of signal intensities and/or ratios will allow changesin labelled probes to be recorded through the reaction or at differentreaction conditions, such as temperatures. The changes are related tothe binding phenomenon between the detection probe and the targetsequence or degradation of the detection probe bound to the targetsequence. The integral of the area under the differential peaks willallow intensity values for the label effects to be calculated.

Screening the mixture for a change in fluorescence provides one or moreassay results, depending on whether the sample is screened once at theend of the primer extension reaction, or multiple times, e.g., aftereach cycle, of an amplification reaction (e.g., as is done in real timePCR monitoring).

The data generated as described above can be interpreted in variousways. In its simplest form, an increase or decrease in fluorescence fromthe sample in the course of or at the end of the amplification reactionis indicative of an increase in the amount of the target present in thesample, e.g., as correlated to the amount of amplification productdetected in the reaction mixture, suggestive of the fact that theamplification reaction has proceeded and therefore the target was infact present in the initial sample. Quantification is also possible bymonitoring the amplification reaction throughout the amplificationprocess. Quantification may also include assaying for one or morenucleic acid controls in the reaction mixture, as described above.

The method of the invention may have a number of uses and applications.One such use may be the preparation of substrates or templates forsequencing or other sequence analysis, such as determining themethylation status of a target ROI. As discussed above, the method mayalso be used for detection, e.g. for identification of a target ROI orfor detection of the presence or absence, or amount or level, of aparticular target ROI in a given sample. Thus the method may be used forthe detection of a target organism, e.g. a pathogen in a sample, whichmay be useful clinically or for research or other purposes, e.g.epidemiological studies or for studying microbial resistance etc.Further the method may find utility in screening for or detecting raremutations, e.g. when screening for minimal residual disease in cancer,particular when combined with sensitive detection strategies such assRCA.

As discussed above, the method of the invention has a number ofadvantages, including for use in such applications, for example a muchreduced sequence error rate. A further advantage is that it does notrequire the correct 5′ and 3′ ends of a probe to be present in order forselection to take place. N-1 and N-2 deletions are commonly present insynthesised nucleic acids, particularly when longer oligonucleotides areused. For circularisation it is also required that the 5′ end of theoligonucleotide to be circularised is phosphorylated. 5′ phosphorylationof probes is a technical hurdle and expensive if large numbers of probesare used. Synthesised nucleic acid probes can often lack a phosphategroup at their 5′ end, which can prevent ligation and circularisation ofa target nucleic acid molecule once it has been selected, for example inthe context of a Selector probe. The present invention thereforebypasses both of these obstacles to the selection of target nucleic acidsequences, reducing the cost associated with ensuring that highlyhomogeneous phosphorylated nucleic acid probes are used.

Advantageously, the present invention can as mentioned above be inhomogenous (non solid phase) or in solid phase-based formats. A selectedtarget nucleic acid can therefore be identified either in situ or in anarray-based assay using any one of a number of techniques known in theart. Use of a solid phase may enable or facilitate washing steps to beincluded.

Since, as mentioned above, the original target molecule is captured inthe circularised target fragment, and not an amplicon or copy thereof,the method permits methylation status to be investigated. Suitablemethods for the analysis of methylation patterns are well known in theart and may include, e.g. mass spectrometry, Methylation-Specific PCR(MSP), bisulfite sequencing (BS-Seq), the HELP assay, DNA microarrays(MeDIP-chip), High Resolution Melt Analysis (HRMA) etc.

The probes, and optionally other components for performing the method ofthe invention may conveniently be provided in kit form.

Accordingly, in a further aspect the invention provides a kit, moreparticularly a kit for selecting a target ROI in a target nucleic acidmolecule, said kit comprising:

(a) a probe of the invention as hereinbefore defined; and optionally oneor more further components selected from:

(b) means for cleaving the cleavage sites created by hybridisation ofthe probe, e.g. one or more restriction enzymes;

(c) means for unfolding a hairpin structure in the probe, e.g. one ormore cleavage enzymes as discussed above;

(d) a ligase enzyme;

(e) one or more gap oligonucleotides;

(f) means for amplification of the circularised target fragment, e.g.one or more amplification primers, (e.g. PCR primers) and/oramplification enzymes (e.g. a polymerase, for example phi29 or anotherstrand displacing polymerase for RCA, or a polymerase suitable for PCR,as known in the art);

(g) means for detecting the circularised target fragment or an ampliconthereof, e.g. one or more detection probes, or labels, or means for afurther detection reaction, e.g. means for performing a sRCA reaction,for example a primer for the sRCA reaction, a template for the sRCAreaction or a padlock probe etc., as described above.

The kit components may be present in separate containers, or one or moreof the components may be present in the same container, where thecontainers may be storage containers and/or containers that are employedduring the assay for which the kit is designed.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, flash drive, etc., on which the information has beenrecorded. Yet another means that may be present is a website addresswhich may be used via the internet to access the information at aremoved site. Any convenient means may be present in the kits.

The invention will be further described in the following non-limitingExamples with reference to the drawings in which:

FIG. 1 illustrates a probe according to the invention and indicates thearrangement of the target binding sites in the probe and the sequencesin the target nucleic acid molecule flanking the target region ofinterest.

FIG. 2 indicates alternative target probes of the present inventioncomprising further elements in addition to the target binding sites. A)Probe with intervening sequences between the first and second, and thirdand fourth target binding sites; B) Probe with an intervening sequencebetween the second and third target binding site and which includes auniversal tag sequence, hybridised to gap oligonucleotide that can actas a circularisation cassette for ligation; C) Probe with interveningsequences between the first and second, second and third, and third andfourth target binding sites. The intervening sequences between the firstand second, and the third and fourth target binding sites may comprisecapture elements; D) Probe with an intervening sequence between thesecond and third target binding site, hybridised to a partiallycomplementary sequence further comprising a sample-specificidentificatory sequence (Sample ID); E) Probe with an interveningsequence between the second and third target binding sites, and with anadditional sequence beyond the first target binding site. Thesesequences can hybridise to form a hairpin structure that can be unfoldedby cleavage adjacent to the first target binding site.

FIG. 3 illustrates the selection method described herein. Probes andgenomic DNA are mixed and undergo denaturation, to allow thehybridisation of three of the target binding sites in the nucleic acidprobe to the target nucleic acid molecule. Next restriction enzymescleave the partially double-stranded nucleic acid molecule to release atarget fragment comprising the target ROI flanked by the first andsecond inner flanking sequences. The ends of the target fragment arethen ligated, templated by the second and third target binding sites.

FIG. 4 indicates the selection method when a nucleic acid probescontains an intervening tag sequence comprising a barcode between thesecond and third target binding sites. The target fragment iscircularised using an additional circularisation cassette (a gapoligonucleotide), complementary to the intervening sequences.

FIG. 5 indicates ways that a probe can be immobilised on a solidsupport. A) A probe with an intervening sequence between the second andthird target binding sites can be immobilised directly on a solidsupport, and a circularisation cassette (gap oligonucleotide)complementary to the intervening sequence is used to aidcircularisation. B) A probe can be immobilised via a sequencecomplementary to an intervening sequence between the second and thirdtarget binding sites. C) A probe can be immobilised via a sequencecomplementary to an intervening sequence between either the first andsecond, or third and fourth target binding sites.

FIG. 6 indicates how a target ROI can be detected in situ in genomicDNA, and RCA products visualised via hybridisation of fluorescentlylabelled oligonucleotides.

FIG. 7 shows a representative probe for use in the method disclosedherein, and illustrates how a nucleic acid probe and target nucleic acidmolecule interact during the various stages of the selection method. A)The probe comprises four target binding sites (labelled TB 1-4), andtarget binding sites 1 and 4 contain sequences capable of creating MboIand HaeIII restriction enzyme recognition sites when hybridised to thetarget molecule. The target nucleic acid molecule comprises a targetregion of interest flanked by first and second inner and outer flankingsequences. B) The probe and target molecule interact to form a partiallydouble-stranded nucleic acid construct. C) Restriction enzyme digestionof the partially double-stranded construct at the MboI and HaeIII sites.D) Releasing target nucleic acid fragment comprising the target ROIflanked by the first and second inner flanking sequences. E)Circularisation of the target fragment templated by the second and thirdtarget binding sites of the probe.

FIG. 8 shows the results of capturing the KRAS target ROI from Example 4using KRAS nucleic acid probes with (T1) and without (T0) poly-Tintervening sequences. The selection method was performed in thepresence and absence of genomic DNA, ModI and HaeIII. Signalamplification by RCA (RCA 1) was also performed, compared to a controlsample (RCA_0).

FIG. 9 shows the results of capturing the SF3B1 target ROI from Example4 using SF3B1 nucleic acid probes with (T1) and without (T0) poly-Tintervening sequences. The selection method was performed in thepresence and absence of genomic DNA, MseI and HaeIII. Signalamplification by RCA (RCA 1) was also performed, compared to a controlsample (RCA_0).

FIG. 10 indicates the sensitivity of the selection method of the presentinvention. 600, 60 and 0 copies of genomic DNA were used and the targetROIs from Example 4 were detected using their respective probes. Signalamplification by RCA (RCA_1) was also performed, compared to a controlsample (RCA_0).

FIG. 11 shows the results of selecting the KRAS target region ofinterest from Example 4 using probes with a barcode sequence between thesecond and third target binding sites. The KRAS_T1, KRAS_InT and KRAS_A2probes were used to select the KRAS target ROI. The selection method wasperformed in the presence and absence of genomic DNA. Signalamplification by RCA (RCA_1) was also performed, compared to a controlsample (RCA_0).

FIG. 12 indicates that the present method can be used to detectmutations within a target ROI. Target sequences were detected usingoligonucleotides labelled with Cy3 and Cy5. Mutant sequences weredetected in approximately 50% abundance in the left hand panel, and aselection of detected amplification products for the mutant sequence arehighlighted with arrows.

FIG. 13 demonstrates that unwanted target-digestion is reduced for theprobes of the present invention, compared with conventionaldouble-stranded selector probes. Loss of signal is only seen when AluI(which cuts double stranded ROI only) is added to the cutting mix in theselector method, but not the method of the present invention.

EXAMPLES

The Examples demonstrate the method of the present invention anddescribe the process of developing probes suitable for selectingsequences from a target nucleic acid molecule. The selection method isshown to result in the target-specific amplification of a nucleic acidproduct, and a number of different nucleic acid probes are shown to workin the method of the invention.

Example 1—Exemplary Nucleic Acid Sequence for the Detection of a Targetof Interest

A KRAS genomic fragment can be detected by the methods of the presentinvention. The sequence of a genomic fragment that can be detected bythe method described herein, and the sequence of a nucleic acid probeused its detection are shown below. The regions of the nucleic acidprobe corresponding to the four target binding sites, and the regions ofthe target nucleic acid molecule corresponding to the flanking sequenceand region of interest are shown in FIG. 7A.

The KRAS genomic fragment (SEQ ID NO:1) has the following sequence (5′ 43′), the target region of interest is shown in bold:

GTCACATTTTCATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAG GTAAATCTTGTTTTAATATG

The nucleic acid probe (SEQ ID NO:2) used to detect the KRAS genomicfragment (KRAS_T0) has the following sequence (5′→3′):

CATATTAAAACAAGATTTACCTCTATTGTTGGATCATACAGTCATTTTCAGCAGGATATTCGTCCACAAAATGAAGCAGGCCTTATAATAAAAATAATGA AAATGTGAC

The interaction between the target binding sites of the nucleic acidprobe and their respective regions of complementarity in the targetnucleic acid molecule is shown in FIG. 7B. FIGS. 7C-7E indicate theprogression of the method of the present invention, which is carried outas shown in FIG. 3. Restriction enzymes MboI and HaeIII are used torelease the target fragment.

Example 2—Genomic Fragment Capture and Amplification by Nucleic AcidProbes

Approximately 1e4 human male genomes (Promega) and 12.5 nM nucleic acidprobes were mixed in 20 μl 0.5×PBS and heated at 95° C. for 20 min,followed by immediate chill on ice. 5 μl cutting and ligation mix wereadded to the target and probe mix reaching final concentrations of 1×CutSmart buffer (NEB), 0.02 U/μl of each restriction enzymes (HaeIII(New England Biolabs (NEB)) and MboI (NEB) for KRAS probes, HaeIII andMseI (NEB) for SF3B1 probes), 0.5 mM NAD (Sigma-Aldrich) and 0.02 U/μlampligase (Epicentre biotechnology). The mix was incubated at 37° C. for30 min and 55° C. for 15 min. One μl of RCA mix was added to 10 μl ofligation products reaching final concentrations of 0.23 μg/μl BSA (NEB),0.57 mM d(A, T, G, C)TP (Thermo Scientific) and 0.05 U/μl phi29 DNApolymerase (Thermo Scientific). The reaction was carried out at 37° C.for 60 min and 65° C. for 15 min. For real-time PCR readout, 40 μl PCRmix were added to RCA products reaching final concentrations of 0.8×PCRbuffer (Invitrogen), 2.5 mM MgCl₂ (Invitrogen), 100 nM of primer pairs(shown in Table 2), 0.5×SYBR (Molecular probes), 0.06 U/μl platinum TaqDNA polymerase (Invitrogen), 0.2 mM d(A, U, G, C)TP (Thermo Scientific),0.002 U/μl UNG (Thermo Scientific). Real time PCR was carried out inStratagene MX3005 PCR machine (Agilent Technologies) using a thermalprofile with an initiation at 95° C. for 2 min followed by 45 cycles of95° C. for 15 sec and 60° C. for 1 min.

TABLE 1List of probes used to detect target nucleic acid regions of interest.SEQ ID Probe Probe NO: name target Probe sequence 2 kRAS_T0 KRASCATATTAAAACAAGATTTACCTCTATTGTTGGATCATACAGTCATTTTCAGCAGGATATTCGTCCACAAAATGAAGCAGGCCTTATAATA AAAATAATGAAAATGTGAC 3kRAS_T1 KRAS CATATTAAAACAAGATTTACCTCTATTGTTGGATCATATTTTTTTTTTTTTTTTTCAGTCATTTTCAGCAGGATATTCGTCCACAAAATGATTTTTTTTTTTTTTTTAGCAGGCCTTATAATAAAAATAATGAAA ATGTGAC 4 KRAS_InT KRASCTATTAAAACAAGATTTACCTCTATTGTTGGATCATATGAATTTCAGCAGGGATACCGGACCAGGTTTCGCCGATTCGAGAACGCAGTGTCATATTCGTCCACAAAATGATGAAAGCAGGCCTTATAATAAAAAT AATGAAAATGTGACGACACTGC 5kRAS_A2 KRAS CTATTAAAACAAGATTTACCTCTATTGTTGGATCATATAAAAAAAAAAAAAAAAAAAAAAAAATCAGTCATTTTCAGCAGGGATACCGGACCAGGTTTCGCCGATTCGAGAACGCAGTGTCATATTCGTCCACAAAATGATGAAAGCAGGCCTTATAATAAAAATAATGAAAATGTGACG ACACTG 6 kRASGACACTGCGTTCTCGAATCGGCGAAACCTGGTCCGGTATC cassette 7 SF3B1_T0 SF3B1GATACCCTTCCATAAAGGCTTTAACACACCAAGGCAGCAATGGACACAGAATCAAAAGATTCGCAATGGCCAAAGCACTGATGGTCCGAA 8 SF3B1_T1 SF3B1GATACCCTTCCATAAAGGCTTTAACACATTTTTTTTTTTTTTTTCCAAGGCAGCAATGGACACAGAATCAAAAGATTCGTTTTTTTTTTTTTTTTCAATGGCCAAAGCACTGATGGTCCGAA

TABLE 2 List of PCR primers used to detect amplification  products.SEQ ID Primer Primer NO: name target Primer sequence  9 kRAS_PCR1 KRASCGTGCCTTGACGATACAGCTAA 10 kRAS_PCR2 KRAS CAAGGCACTCTTGCCTACG 11SF3B1_PCR SF3B1 GATTCTGTGTCCATTGCTGC 1 12 SF3B1_PCR SF3B1GAGTTGCTGCTTCAGCCAAG 2

TABLE 3 List of target nucleic acid molecules. SEQ ID Target NO: nameTarget sequence  1 kRAS GTCACATTTTCATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGG AGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATG ATCCAACAATAGAGGTAAATCTTGTTTTAATATG 13SF3B1 GTCTTGTGGATGAGCAGCAGAAAGTTCGGACCATCAGTGCTTTGGCCATTGCTGCCTTGGCTGAAGCA GCAACTCCTTATGGTATCGAATCTTTTGATTCTGTGTTAAAGCCTTTATGGAAGGGTATCCGCCAACA CAGAGGAAAG

The KRAS_T0 and KRAS_T1 nucleic acid probes shown in Table 1 were usedto select the target region of interest from the KRAS genomic fragmentshown in Table 3. The target region of interest is shown in bold. TheKRAS_T1 probe comprised the same target binding sites as the KRAS_T0probe, but further comprised a poly-T intervening sequence between itsfirst and second, and third and fourth target binding sites.

Positive signals were only seen for the amplification product after PCRamplification when genomic DNA and both the MboI and HaeIII restrictionenzymes are present in the reaction sample. Where only one of therestriction enzymes or genomic DNA was not present, near baselinesignals were detected. Signals from reactions with RCA amplifications(RCA_1) were ˜1000-fold increased compared to reactions with no RCA(RCA_0), indicating successful circularisation of target fragments afterenzymatic cleavage. No significant difference was observed for theprobes with and without the poly-T intervening sequences. The results ofthe amplification of the KRAS target region of interest are shown inFIG. 8. A detection efficiency of 56% was estimated for the KRAS_T1probe, assuming that approximately 1e4 genomes were present in eachassay with 40% sampling efficiency throughout the protocol. A 1 hour RCAproduces ˜1000-fold amplification at 37° C. and Ct 40 corresponds to 1copy of a DNA amplicon in a PCR reaction. Error bars are the standarddeviation for duplicate measurements.

The SF3B1_T0 and SF3B1_T1 nucleic acid probes shown in Table 1 were usedto select the target region of interest from the SF3B1 genomic fragmentshown in Table 3. The target region of interest is shown in bold. TheSF3B1_T1 probe comprised the same target binding sites as the SF3B1_T0probe, but further comprised a poly-T intervening sequence between itsfirst and second, and third and fourth target binding sites.

Positive signals were only seen for the amplification product after PCRamplification when genomic DNA and both the MseI and HaeIII restrictionenzymes are present in the reaction sample. Where only one of therestriction enzymes or genomic DNA was not present, near baselinesignals were detected. Signals from reactions with RCA amplifications(RCA_1) were ˜1000-fold increased compared to reactions with no RCA(RCA_0), indicating successful circularisation of target fragments afterenzymatic cleavage. No significant difference was observed for theprobes with and without the poly-T intervening sequences. The results ofthe amplification of the SF3B1 target region of interest are shown inFIG. 9. A detection efficiency of 3% was estimated for the SF3B1_T1probe. Error bars are the standard deviation for duplicate measurements.

Example 3—Selection of Low Copy-Number Target Nucleic Acid Molecules

The method of the present invention was used to detect a low copy numberof genomic DNA targets in order to determine the sensitivity of themethod of the present invention. 60 or 600 genomic targets were added tothe test reactions, and both KRAS and SF3B1 genomic fragments weredetected according to the method outlined in Example 4.

Both target regions of interest were detectable even at the 60 genomeslevel, indicating that the present method is highly sensitive and candetect a low copy-number of target nucleic acid molecules. Overall nosignificant differences were observed between the efficiency ofdetection for the probes with, and without the poly-T interveningsequences. The results of the low copy-number detection of the targetnucleic acid fragments is shown in FIG. 10. A detection efficiency of2.5% of 600 genomes was estimated for the SF3B1_T0 probe. Error bars arethe standard deviation for duplicate measurements.

Example 4—Effect of a Barcode Sequence on Amplification Efficiency

Nucleic acid probes with and without an barcode sequence between thesecond and third target binding sites (i.e. a barcode sequence) wereused to determine the effect that the barcode sequence had onamplification efficiency. The KRAS genomic fragment was detectedaccording the method outlined in Example 4 using the KRAS_T1, KRAS_InT,and KRAS_A2 probes shown in Table 1. The KRAS_InT and KRAS_A2 probescomprise a barcode sequence between the second and third binding sitesin the probe. As the KRAS_T1 probe had already been shown to be aseffective as the KRAS_T0 probe for selecting the KRAS target fragment,it was used as the positive control probe for this experiment.

No significant loss of signal was observed when using probes containinga barcode sequence (i.e. KRAS_InT and KRAS_A2) in comparison to theKRAS_T1 probe. No significant difference in signals was observed whenprobes with a polyT (kRAS_InT) insertion were used in comparison toprobes with a polyA (kRAS_pA2) insertion. The results of theamplification of the KRAS target region of interest are shown in FIG.11. A detection efficiency of ˜2% was estimated for KRAS_InT probes.Error bars are the standard deviation for duplicate measurements.

Example 5—Mutation Detection by Nucleic Acid Probes and superRCA Readout

KRAS G12A Genomic DNA reference standards (wildtype and mutant (50%))were purchased from Horizon Diagnostics and the region of interests(ROI) were enriched by PCR using primers reaching out fragments forcomplete binding of CutLig probes. Approximately 1.5e9 enrichedfragments were mixed with 100 fold excess of probes and processedaccording to the protocol described in Example 4. One μl of SuperRCApadlock mix was added to 15 μl RCA mix reaching final concentrations of0.05 mM ATP (Thermo Scientific), 100 nM of each padlock and 0.04 WeissU/μl T4 DNA ligase (Thermo Scientific). The reaction was incubated at37° C. for 30 min, followed by addition of 1.2 μl 2^(nd) RCA mixreaching final concentrations of 1 mM dNTP, 500 nM 2^(nd) RCA primer,100 nM of each Cy3 or Cy5 labelled detection oligonucleotides and 0.1U/μl phi29 DNA polymerase. The RCA reaction was carried out at 37° C.for 120 min. Four μl of RCA products were deposited on a poly-L-lysinecoated glass slide (Sigma-Aldrich), followed by image acquisition using20× objective of an Axioplan II epifluorescence microscope (Zeiss) withexcitation and emission filters for Cy3 and Cy5 and exposure time of1000 ms.

SuperRCA products were generated for both the wildtype and mutant DNAsequences. Mutant amplification product was detected in the red channel,and wild-type amplification product was detected in the green channel.The mixture of the mutant and wild-type DNA sequences were detected inthe red and green channels in equal number, whereas the wild-typesequences were only detected in the green channel. FIG. 12 shows theresults of the mutant detection in the KRAS gene.

Example 6—Demonstration of Reduced Unwanted-Target-Digestion UsingNucleic Acid Probes

A direct comparison of the effect of AluI on reducing the signalobtained when selecting a target nucleic acid sequence was made betweenthe known “selector” method of selecting a target region of interest asdescribed in U.S. Pat. No. 7,883,849, and the method disclosed in thepresent invention.

For the method of the invention (CutLig approach), approximately 3e6human male genomes (Promega) were incubated with 25 nM probes in 20 μl0.5×PBS and heated at 95° C. for 20 min, followed by immediate chill onice. 27 μl cutting mix containing 1× CutSmart buffer, 0.05 U/μl HaeIII,0.05 U/μl MboI was added to 3 μl target and probe mix, followed byincubation at 37° C. for 30 min and 85° C. for 20 min. For Selectorapproach, approximately 3e6 human male genomes were incubated in 30 μl1× CutSmart buffer, 0.05 U/μl HaeIII, 0.05 U/μl MboI at 37° C. for 30min and 85° C. for 20 min. 2 μl of the digested products were incubatedwith 18 μl 2.5 nM probes in 0.5×PBS at 95° C. for 20 min, followed byimmediate chill on ice.

AluI was added to the cutting mix in both approaches for digestion ofdouble stranded target region of interest. 3 μl of the products fromeach approach were then incubated in 30 μl of 1× ampligase buffer, 0.02U/μl ampligase at 55° C. for 15 min. 6 μl of the ligation products wereRCA amplified in 30 μl of 1×EINAR buffer (50 mM KAc, 20 mM Tris-HAc pH7.6, 3 mM MgAC₂), 0.2 μg/μl BSA, 0.25 mM d(A, T, G, C)TP and 0.02 U/μlphi29 DNA polymerase at 37° C. for 60 min and 65° C. for 15 min. 10 μlRCA products were added to 15 μl PCR mix reaching final concentrationsof 1×EINAR buffer, 100 nM PCR primers, 0.5×SYBR, 0.06 U/μl platinum TaqDNA polymerase 0.2 mM d(A, U, G, C)TP, 0.002 U/μl UNG. Real time PCR wascarried out in Stratagene MX3005 PCR machine (Agilent Technologies)using a thermal profile with an initiation at 95° C. for 2 min followedby 45 cycles of 95° C. for 15 sec and 60° C. for 1 min.

AluI cuts double-stranded DNA at the sequence AGCT, which is located attwo separate locations within the target region of interest in the KRASgene. FIG. 13 shows the effect of AluI on the level of amplificationproduct for both the selector method and the method of the presentinvention. The extent of cleavage of the DNA product of the presentinvention is substantially lower than that seen for the selector method,as the target region of interest is single-stranded during therestriction digestion step of the method of the present invention (asthe nucleic acid probes of the present invention do not comprise asequence capable of binding to the target region of interest). Thisdemonstrates that a particular restriction enzyme may be used in themethods of the present invention, even when the target region ofinterest contains the recognition sequence for that particularrestriction enzyme, without significantly degrading the target region ofinterest or affecting the sensitivity of the selection method.

In the methods of the prior art it was previously necessary to avoidusing restriction enzymes which had recognition sequences within thetarget region of interest, as the double-stranded products producedwould be degraded. In contrast to this, in the method of the presentinvention such a restriction enzyme or enzymes may be used, as thetarget region of interest is not degraded. This is of particularinterest in the multiplexing aspect of the present invention, where itmay be desirable to use many different restriction enzymes whenselecting a plurality of target regions of interest, as it is no longernecessary to avoid using a particular restriction enzyme due to thepresence of a particular sequence within one or more of the plurality oftarget regions of interest. Thus a larger number of restriction enzymesmay be used in the multiplexed selection of a plurality of targetnucleic acids than was previously possible, and thus the presentinvention enables a higher degree of multiplexing to take place.

1. A method of selecting a target region of interest (ROI) in a targetnucleic acid molecule, said method comprising: (a) providing a probecomprising in the following order four target-binding sites capable ofhybridising to complementary binding sites in the target molecule, whichcomplementary binding sites flank the target ROI, as follows: (i) afirst target-binding site, complementary to a first outer flankingsequence flanking a first side of the ROI in the target molecule; (ii) asecond target-binding site, complementary to a second inner flankingsequence flanking the other side of the ROI on the target molecule;(iii) a third target-binding site, complementary to a first innerflanking sequence flanking the first side of the ROI in the targetmolecule; (iv) a fourth target-binding site, complementary to a secondouter flanking sequence flanking the other side of the ROI in the targetmolecule; such that only one of the second and third binding sites areable to hybridise to their respective complementary binding site in thetarget molecule when said first, fourth and the other of second andthird binding sites have hybridised; wherein the first and fourthbinding sites comprise a sequence capable of creating a cleavage sitewhen hybridised to the target molecule; (b) contacting the probe withthe target molecule and allowing the first, fourth and one of the secondand third target binding sites to hybridise to their respectivecomplementary binding sites in the target molecule, wherein the targetmolecule is at least partially single stranded including in the regioncomprising the four complementary binding sites, such that when saidprobe has bound a partially double-stranded construct is createdcomprising a loop in the probe strand comprising the second or thirdtarget binding site which did not hybridise to the target molecule and aloop in the target molecule strand comprising the ROI and thecomplementary binding site which is complementary to the second or thirdtarget binding region of the probe which did not hybridise; (c) cleavingthe probe/target molecule construct at the cleavage sites created byhybridisation of the first and fourth target-binding sites thereby torelease a target fragment comprising the ROI flanked by the first andsecond inner flanking sequences which are complementary to the third andsecond target binding regions of the probe, one of which is hybridisedto its complementary binding site in the cleaved probe; (d) allowing thesecond or third target-binding site of the probe which did not hybridisein step (b) to hybridise to its complementary binding site in the targetfragment, thereby to bring the ends of the target fragment intojuxtaposition for ligation, directly or indirectly, using the cleavedprobe as ligation template; (e) ligating the ends of the target fragmentdirectly or indirectly to circularise the target fragment; (f)amplifying or separating the circularised target fragment, thereby toselect the ROI.
 2. The method of claim 1, wherein a plurality of probesare used to select a plurality of target regions of interest, whereineach probe is designed to select a different target region of interest.3. The method of claim 1, wherein a single probe is used to select aplurality of different target regions of interest within the same targetmolecule derived from a variety of sources.
 4. The method of claim 1,wherein the first and fourth target binding sites are situated at theends of the probe.
 5. The method of claim 1, wherein the four bindingsites are immediately adjacent to one another.
 6. The method of claim 1,wherein the probe further comprises one or more intervening sequencesbetween any two of the binding sites.
 7. The method of claim 6, whereinthe intervening sequence is a capture sequence.
 8. The method of claim7, wherein the capture sequence hybridises to a cognate complementarybinding site provided on a solid surface.
 9. The method of claim 7,wherein the capture sequence or a complementary oligonucleotidehybridised thereto is attached to an affinity binding moiety.
 10. Themethod of claim 7, wherein a capture sequence is provided between thefirst and second and/or between the third and fourth binding sites. 11.The method of claim 6, wherein the intervening sequence is or comprisesa tag sequence or a complement thereof, wherein the tag sequence isselected from a detection sequence or an identification sequence elementor a binding site for a primer or detection probe.
 12. The method ofclaim 11, wherein the tag sequence is an identification element for thetarget ROI or a sample identification sequence.
 13. The method of claim1, wherein the second and third binding sites are immediately adjacentto one another and the ends of the target fragment are ligated directlyto one another.
 14. The method of claim 6, wherein an interveningsequence lies between the second and third binding sites, therebycreating a gap between the respective ends of the target fragment whenthey are both hybridised to the second and third binding sites in step(d), and wherein the gap is filled prior to ligation by one or more gapoligonucleotides which hybridise in the gap between the ends of thetarget fragment, or by gap-fill extension of the 3′ end of thehybridised target fragment using a polymerase, such that the targetfragment and any gap oligonucleotides if present may be ligated into acircular molecule comprising the target fragment.
 15. The method ofclaim 14, wherein the gap oligonucleotide(s) comprise(s) a tag sequencecomplementary to a tag sequence complement in the intervening sequence.16. The method of claim 14, wherein the gap oligonucleotide comprises aregion which is not complementary to the intervening sequence, andwherein the region of non-complementarity comprises a tag sequence. 17.The method of claim 14, wherein the gap oligonucleotide comprises adetection sequence or an identification sequence element, or a bindingsite for an amplification primer or a detection probe.
 18. The method ofclaim 17, wherein the amplification primer is a universal amplificationprimer.
 19. The method of claim 14, which is performed in multiplexusing a plurality of different probes and wherein the gapoligonucleotide for each probe comprises the same tag sequence,preferably the same primer binding site.
 20. The method of claim 14,wherein the gap oligonucleotide is pre-hybridised to the probe prior tocontacting the probe with the target nucleic acid molecule.
 21. Themethod of claim 14, wherein the gap oligonucleotide is separatelyprovided at the same time or after contacting the probe with the targetmolecule.
 22. The method of claim 6, wherein the probe comprises ahairpin structure containing one or more binding sites, such that theyare not available for hybridisation to the target molecule until theprobe is activated by unfolding the hairpin structure, and wherein themethod comprises a further step of activating the probe by unfolding thehairpin structure by disruption of the duplex of the hairpin or bycleavage in or near the loop prior to or after contacting the probe withthe target molecule.
 23. The method of claim 22, wherein the stem of thehairpin is formed between an intervening sequence between the second andthird binding sites of the probe, and an additional sequence provided atthe end of the first binding site, and wherein cleavage of the loop ator near to the junction between the first target binding site and theadditional sequence in the stem duplex releases the first and secondbinding sites.
 24. The method of claim 23, wherein cleavage is enzymaticcleavage.
 25. The method of claim 1, wherein the cleavage sites createdin step (b) are restriction sites.
 26. The method of claim 1 whereinamplification of the circularised target fragment is by PCR or RollingCircle Amplification (RCA).
 27. The method of claim 26, whereinamplification is by RCA, and wherein amplification further comprises asecond round of RCA.
 28. The method of claim 1 further comprising a stepof detecting and/or analysing the circularised target fragment or anamplicon thereof.
 29. The method of claim 28, wherein the amplifiedproduct is detected by hybridising a detection probe labelled with adirectly or indirectly detectable label to the amplification product.30. The method of claim 29, wherein the detection label is a fluorescentlabel.
 31. The method of claim 28, wherein the target fragment oramplicon thereof is detected or analysed by sequencing.
 32. A probe foruse in the method of claim 1, said probe comprising in the followingorder four target-binding sites capable of hybridising to complementarybinding sites in the target molecule, which complementary binding sitesflank the target ROI, as follows: (i) a first target-binding site,complementary to a first outer flanking sequence flanking a first sideof the ROI in the target molecule; (ii) a second target-binding site,complementary to a second inner flanking sequence flanking the otherside of the ROI on the target molecule; (iii) a third target-bindingsite, complementary to a first inner flanking sequence flanking thefirst side of the ROI in the target molecule; (iv) a fourthtarget-binding site, complementary to a second outer flanking sequenceflanking the other side of the ROI in the target molecule; such thatonly one of the second and third binding sites are able to hybridise totheir respective complementary binding site in the target molecule whensaid first, fourth and the other of second and third binding sites havehybridised; and wherein the first and fourth binding sites comprise asequence capable of creating a cleavage site when hybridised to thetarget molecule.
 33. (canceled)
 34. A kit for selecting a target regionof interest in a target nucleic acid molecule, said kit comprising: a) aprobe as defined in claim 32 and optionally one or more componentsselected from: b) means to cleaving the cleavage sites created byhybridisation of the probe; c) means for unfolding a hairpin structurein the probe; d) a ligase enzyme; e) one or more gap oligonucleotides;f) means for amplification of the circularised target fragment; and g)means for detecting the circularised target fragment or an ampliconthereof.